Principles of plant biotechnology Lecture note.pptx

Lecture note

1. Plant Biotechnology Biot 3093 1 School of Biological Sciences and Biotechnology Haramaya University

2. Methods of Biotechnology Gene Transfer: Vectors Restriction Enzymes Polymerase Chain Reaction DNA Ligase and Cloning The Accomplishment and Opportunities of Plant Biotechnology Genetic Engineering: Trans- genic Plants Advantages and Disadvantages of Genetic Engineering Brief History of The First GM crops Genomics and Proteomics Topics to be covered 2 School of Biological Sciences and Biotechnology Haramaya University

3. Presentation Objectives By the end of this presentation, listeners should know how to: Define plants biotechnology Identify the methods of gene transfer Explain the polymerase chain reaction Identify some genetically engineered crops Know the contribution of trans-genic plants to human health and nutrition Have knowledge of genomics and proteomics 3 School of Biological Sciences and Biotechnology Haramaya University

4. What is Plant Biotechnology? Biotechnology is a field of applied biology that involves the use of living organisms, cells, bioprocesses, biomaterials in different fields of humans’ uses such as engineering, technology, medicine and agriculture. Plant Biotechnology is a collection of technologies that including a using of plant cells, bioprocesses, macro-& micromolecules for manufacturing/ produce specific products. 4 School of Biological Sciences and Biotechnology Haramaya University

5. Historical Overview of Plant Biotechnology Matthias Schlieden and Theodor Schwan proposed the concept of cell theory in the 19th century Gottlieb Haberlandt , a German botanist proposed the plant tissue culture. 5 School of Biological Sciences and Biotechnology Haramaya University Gottleib Haberlandt

6. Historical Overview cont’d 1902 – Haberlandt – The Concept 1920s – Knudson – Simple Orchid Germination – First commercial use 1930s – Thimann & Went – Auxin 1930s – White/ Gautheret / Nobecourt –Root Cultures 1950s – Skoog’s group – Cytokinins , – The discovery of the structure of DNA by Crick and Watson 1960s – Morel-Orchid microprop agation , thermotherapy 1970’s – G enetic engineering took off 1990s – by Calgene – genetically engineered potatoes 6 School of Biological Sciences and Biotechnology Haramaya University

7. WHAT IS TISSUE CULTURE? PLANT TISSUE CULTURE -Technique of growing plant cells, tissues, and organs in an artificially prepared nutrient medium under aseptic condition. REQUIREMENT OF TISSUE CULTURE : Appropriate tissue ( explant ) A suitable growth medium ( Some of the common media are MS, LS, Gamborg B5, White’s, Hellar for haploids etc) Growth regulators Aseptic (sterile) conditions Frequent subculturing : . 7 School of Biological Sciences and Biotechnology Haramaya University

8. True-to-type clones A single explant can be multiplied into several thousand Year-round production R are and endangere d plants can be cloned safel y To produce virus free plants Long-term germplasm storage with ‘tissue banks’ Plant cultures e asier t o export than are soil-grown plants Production of difficult-to-propagate species Worldwide industryin multi billion ADVANTAGES OF PLANT TISSUE CULTURE TECHNIQUES

9. FUNDAMENTAL AB I L I T I ES OF PLANTS HOW CAN A PLANT CELL OR TISSUE DEVELOP? Therefore, tissue can be regenerated from explants such as cotyledons, hypocotyls, leaf, ovary, protoplast, roots, anthers, etc .

10. Plant Tissue Culture Media A substrate on which in-vitro propagated explants are placed for proper nourishment and growth a mixture of chemical constituents in an appropriate ratio, suitable to support the in vitro growth and development of plants Appropriate media selection and preparation - a vital factor that must be put into consideration for a successful tissue culture. School of Biological Sciences and Biotechnology Haramaya University 10

11. Commonly used types of plant tissue culture media MS ( Murashige and Skoog , 1962) SH ( Shenck and Hildebrandt) B 5 ( Gamborg’s ) WPM (Woody plant medium). Generally, nutrient media comprises of: Water Inorganic nutrients Carbon sources Vitamins Growth regulators Organic supplements: myoinositol , and glycine Other components include: Agar (medium matrix) Charcoal Amino acids or amides Antibiotics Ascorbic Acid ( used as antioxidant to stop tissue browning) School of Biological Sciences and Biotechnology Haramaya University 11

12. Water Serves as the solvent in which all the added components are dissolved Only de-ionized or de-mineralized water is acceptable It is also important to use freshly de-ionized water. Therefore, prolonged storage should be discouraged Inorganic nutrients Macronutrient elements Micronutrient elements EDTA is usually added prior to the addition of Iron in order to ensure that the ionic state of the Iron doesn’t change i.e from Fe 2+ to Fe 3+ School of Biological Sciences and Biotechnology Haramaya University 12

13. Stock solutions for Murashige and Skoog (MS) medium Inorganic nutrients STOCK 1 STOCK 2 Constituents Amount g/ lL KI 0.166 H2BO3 1.240 MnSO4 4H2O 4.460 ZnSO4 7H2O 0.050 CuSO4.7H2O 0.005 COCl2.6H2O 0.005 School of Biological Sciences and Biotechnology Haramaya University 13 Constituents Amount (g/L) NH 4 NO3 33 KNO3 38 Cacl2 2H2O 8.8 MgSO4 7H2O 7.4 KH2PO4 3.4 STOCK 3 Constituents Amount (g/L) FeSO4.7H2O 0.278 Na2EDTA2H2O 0.373

14. Carbon Sources All cultured cells utilize sucrose and glucose equally well. Sucrose or glucose at 2-3%w/v is commonly used in cell culture (20-30g/L). Other carbohydrate source such as fructose and starch can also be used in protoplast culture while much higher levels may be used for embryo or anther culture. However, the hexitols e.g. myo-inositol , have been found to be very important in tissue culture (@ 0.1g/L) School of Biological Sciences and Biotechnology Haramaya University 14

15. Vitamins Vitamins are needed in small amounts due to their catalytic function in enzyme systems. These include: Thiamine (B1) Nicotinic Acid (B3) Pyridoxine (B6) Amino-benzoic Acid Vitamin B12 Folic Acid Biotin VITAMIN STOCK Constituents Amount (g/L) Nicotinic acid 0.1 Pyridoxin HCL 0.1 Thiamin HCL 0.1 Glycine 0.2 School of Biological Sciences and Biotechnology Haramaya University 15

16. Growth Regulators Growth regulators – hormones - initiator of an internal stimuli within the plant system. In a culture medium – can significantly determine the response rate of a tissue culture system 3 types of PGRs are usually employed: Auxins - Promote cell division, root initiation and elongation. Cytokinins - Promote cell division, shoot elongation, multiplication and morphogenesis Gibberellins-promote height in plant. The type and concentration of plant growth regulators used will vary according to the cell culture purpose COMMON PGRs USED IN PLANT T.C. Auxins . Abbreviation Abscisic acid (ABA) Napthalene acetic acid (NAA) 2,4-dichlorophenoxyacetic acid (2,4-D) Indole-3-acetic acid (IAA) Indole-3-butyric acid (IBA) Cytokinins . 6-benzyl-aminopurine (BAP) 6-furfuryaminopurine kinetin Zeatin zeatin Gibberellins Gibberellic acid (GA3) School of Biological Sciences and Biotechnology Haramaya University 16

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18. Preparation of Growth Regulator stocks Auxin stock - weigh out 0.01g auxin into a beaker, add few drops of a base ( NaOH or KOH) to dissolve crystals, - make up to 100ml vol. with distilled water in a volumetric flask. IAA stock - prepared fresh - degraded by light within several hours to a few days. Cytokinins stock - prepared in a similar way to auxin stock except that crystals are dissolved with acid ( HCl ) along with few drops of water. Gentle heating - dissolve crystals. De-ionized water -added quickly - to avoid the crystals falling out of solution. Make up to final volume in100ml volumetric flask. Cytokinins stocks can be stored for several months in the refrigerator Gibberellin : Stock solution of GA3 can be prepared by dissolving in water and adjusting the pH to 5.7, GA3 is not thermostable and 20mins at 114 ° C reduces its activities by more than 90% School of Biological Sciences and Biotechnology Haramaya University 18

19. Vitamins Vitamins have a catalytic function in enzyme reactions. The vitamin required by plant cells is thiamine (B1) Others include: Nicotinic acid (B3) and pyridoxine (B6). The vitamins stock is prepared by dissolving in water. Most vitamins are soluble in water. Vitamins stocks are best stored in a freezer. Carbohydrates Green cells in culture are generally not photosynthetically active and require a carbon source. Sucrose (table sugar) is commonly used in cell culture – 20-30g. Myo-inositol is another carbohydrate that is commonly added- 0.1g. School of Biological Sciences and Biotechnology Haramaya University 19

20. Gelling agents - Used to create a semi-solid or solid media. Agar - most commonly used gelling agent. When agar is mixed with liquid, it forms a gel that melts at about 100 ° C and solidifies at 45 °C . Agar does not react with any component of the medium It is not digested by enzymes from plant tissues All agar contain impurities which usually do not interfere with culture response. However, the concentration of agar is also critical to plant response in culture (5-7g). Natural Complexes Undefined supplements such as Coconut milk, Yeast extract, fruit juice and fruit pulps May supply amino-acids, vitamins, plant growth regulators and /or secondary plant metabolites N.Cs were initially used earlier in P.T.C. history, when growth requirement were less defined. But now, they are used only when no combination of defined components supports growth. - Their disadvantages are that the important compounds in them are not known, and may vary greatly from batch to batch School of Biological Sciences and Biotechnology Haramaya University 20

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23. Basic Laboratory Procedures Involved in Media Preparations School of Biological Sciences and Biotechnology Haramaya University 23 Nutrients Standard medium conc. (mg/L) Strength of stock solution Stock solution conc. (g/L) Amount required (g/L) for 500 ml and 100 ml of stock solution, respectively Volume needed in 1L medium Volume needed in 500mL medium Calcium chloride anhydrous (CaCl 2 .2H 2 O) 440 Stock A (X10) 4.4 2.2 100ml 50 Potassium nitrate (KNO3) 1900 19.0 9.5 Ammonium nitrate (NH4NO3) 1650 16.5 8.25 Potassium phosphate monobasic (KH2PO4) 170 1.7 0.85 Magnesium sulfate (MgSO47H2O) 370 Stock B (X10) 3.7 1.85 100ml 50ml Manganese sulfate (MnSO4.4H2O) 22.3 0.223 0.1115 Zinc sulfate (ZnSO 4 .7H 2 O) 8.6 Stock C (X100) 0.86 0.083 10ml 5ml Boric acid (H3BO3) 6.2 0.62 0.062 Potassium iodide (KI) 0.83 0.083 0.0083 Cupric sulfate (CuSO 4 .5H 2 O) 0.025 0.0025 0.00025 Molybdic acid (Na 2 MoO 4 .2H 2 O) 0.25 0.025 0.0025 Cobalt chloride (CoCl 2 .6H 2 O) 0.025 0.0025 0.00025 Na2EDTA.2H2O 37.3 Stock D (X100) 3.73 0.373 10ml 5ml Ferrous sulfate (FeSO 4 .7H 2 O) 27.8 2.78 0.278 Glycine 2.0 Stock E (X100) 0.2 0.02 10ml 5ml Nicotinic Acid 0.5 0.05 0.005 Thiamine-HCl 0.1 0.01 0.001 Pyridoxine-HCl 0.5 0.05 0.005 Myo-inositol (add directly into medium) 100 0.1g 0.05g

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25. Culturing ( micropropagating ) Plant Tissue - the steps Stage 0 – Selection & preparation of the mother plant sterilization of the plant tissue takes place Stage I  - Initiation of culture explant placed into growth media Stage II - Multiplication explant transferred to shoot media; shoots can be constantly divided Stage III - Rooting explant transferred to root media Stage IV - Transfer to soil explant returned to soil; hardened off

26. PROCESS OF PLANT TISSUE CULTURE Selection of explant from mother plant Parent plant Inoculation Light &warmth for shoot initiation Shoots are transferred to rooting media(higher auxin conc ) Shoot formation Callus induction Plants are kept for hardening in green house ,sold to a nursery and then potted up. The cells first forms a mass of undifferentiated cells called callus

27. Tissue culture plants sold to a nursery & then potted up The rooted shoots are potted up ( deflasked ) and ‘hardened off ’ This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss.

28. TYPE S OF PLANT TISSUE CULTURE TECHNIQUES Micropropagation Culture of i ntact plants (Seed orchid culture) Embryo culture (embryo rescue) Organ culture :Micropropagation A. Organogenesis in solid or semi solid medium 1. Meristem and shoot tip culture 2. Bud culture 3. Root culture 4 . Leaf culture 5 . A nther culture B. Somatic embryogenesis C. Organogenesis and somatic embryogenesis in bioreactors D. In vitro micrografting E. Thin cell layer technology (TCLs) F. Photoautotrophic culture 4. Callus culture 5. Cell suspension and single cell culture 6. Protoplast culture , somatic hybridization

29. FACTORS AFFECTING TISSUE CULTURE Growth Media Minerals, Growth factors, Carbon source, Hormones Environmental Factors Light, Temperature, Photoperiod, Sterility, Media Explant Source Usually, the younger, less differentiated explant , the better for tissue culture Different species show differences in amenability to tissue culture In many cases, different genotypes within a species will have variable responses to tissue culture; response to somatic embryogenesis has been transferred between melon cultivars through sexual hybridization

30. Tissue Culture Applications Micropropagation Germplasm preservation Somaclonal variation Haploid & dihaploid production In vitro hybridization – protoplast fusion Embryo rescue Synthetic seed production Production of secondary metabolites Biotransformation

31. Plants’ Secondary Metabolites School of Biological Sciences and Biotechnology Haramaya University 31

32. Primary Metabolites School of Biological Sciences and Biotechnology Haramaya University 32

33. Plants’Secondary Metabolites Secondary metabolites are those metabolites which are often produced as by product of growth development of plants , have no function in growth (although they may have survival function), are produced by certain restricted taxonomic groups of microorganisms, have unusual chemicals structures, and are often formed as mixtures of closely related members of a chemical family. A metabolic intermediate or product, found as a differentiation product in restricted taxonomic groups, not essential to growth and the life of the producing organism, and biosynthesis from one or more general metabolites by a wider variety of pathways than is available in general metabolism. Secondary metabolites are not essential for growth and tend to be strain specific. They have a wide range of chemical structures and biological activities. They are derived by unique biosynthetic pathways from primary metabolites and intermediates. Biochemical pathways that are not necessary for growth or reproduction of an organism, but which can be demonstrated genetically, physiologically or biochemically. School of Biological Sciences and Biotechnology Haramaya University 33

34. Plant Secondary Metabolites The ability to synthesize secondary metabolites has been selected through the course of evolution in different plant lineage when such compounds address specific needs. Floral scent volatiles and pigments have evolved to attract insect pollinators and thus enhance fertilization. To synthesize toxic chemical has evolved to ward off pathogens and herbivores or to suppress the growth of neighboring plants . Chemicals found in fruits prevent spoilage & act as signals (in the form of color, aroma, and flavor) of the presence of potential rewards (sugars, vitamins and flavor) for animals that eat the fruit and thereby help to disperse the seeds. Other chemicals serve cellular functions that are unique to the particular plant in which they occur (e.g. resistance to salt or drought ).

35. Types of Secondary Metabolites These are highly numerous in number, chemically diverse in nature and belong to three groups. 1. Polyketides : Isoprenoids or Terpenes , e.g., rubber, steroids, essential oils, carotenoid pigments. 2. Nitrogen containing compounds , e.g., alkaloids, glucosinolates , glycosides, non-protein amino acids. 3. Phenolic compounds , e.g., lignin, tannins, coumarins , aflatoxins , Tannins, flavonoids . 4.Sulphur containing secondary metabolites: Glutathione (GSH ) , Amino acid-derived glucosinolates  (GSLs ) Phytoalexins , Thionins , Defensins , Allinin Polyketides are formed by the linear combination of acetate units derived from the “building block” acetyl co- enzyme A. School of Biological Sciences and Biotechnology Haramaya University 35

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42. 4.Sulphur containing secondary metabolites They include: GSH GSL Phytoalexins Thionins Defensins Allinin They linked with the defence of plants against microbial pathogens.

43. Phytoalexins Phytoalexins are synthesized in response to bacterial or fungal infection. They help in limiting the spread of the invading pathogens by accumulating around the site of infection.

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45. Significance of Secondary Metabolites Secondary metabolites attract animals for pollination and seed dispersal. The plants used in their defence against herbivores and pathogens. They act as agents of plant-plant competition. They are used in making drugs, insecticides, flavours, pigments, scents, rubber, spices and other industrial materials like gums, resins for human welfare. School of Biological Sciences and Biotechnology Haramaya University 45

46. Applications of Secondary Metabolites: Human has been dependent on the plant products, besides the supply of food from plants. These plant products, mostly the secondary metabolites include pharmaceuticals, flavours , perfumes, agrochemicals, insecticides and raw materials for industries. School of Biological Sciences and Biotechnology Haramaya University 46

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48. Discovery of Plant Growth Regulators The discovery of major Plant growth regulators began with observations of plant movements by Charles Darwin (1884) and his son, Francis Darwin. They observed the growth of coleoptiles of canary grass ( Phalares canariensis ) towards the light source- phototropism . Followed by a series of experiments, they concluded the presence of a transmittable substance that influences the growth of canary grass towards the light. That transmittable substance was what we know as auxin which was isolated later by F.W. Went (1928). Effect of Auxin on Plant Growth Later many scientists discovered and isolated different plant growth regulators. Gibberellins or gibberellic acid was formerly found in uninfected rice seedlings and was reported by E. Kurosawa. Skoog and Miller discovered another growth promoting substance named kinetin, which is now known as cytokinin.

49. 6/12/2024 49 Plant Hormones Plant hormones classification: The five classical hormones: Auxin Cytokinin Gibberellin Abscisic acid Ethylene Additional substances considered as plant Hormones: Polyamines Jasmonates Salicylic acid Brassinosteroids Signal peptides

50. Types of Plant Growth Regulators Generally, there are five types of plant hormones namely, auxin, gibberellins (GAs), cytokinins , abscisic acid (ABA) and ethylene. In addition to these, there are more derivative compounds, both natural and synthetic like CCC, which also act as plant growth regulators. Auxins , Gibberellins, and Cytokinins are grouped into Plant growth promoters while Abscisic acid, Ethylene and CCC are grouped into Plant growth inhibitors . Ethylene can be grouped either into the promoters or into the plant inhibitors.

51. Plant Growth Promoters Auxins : The first phytohormones to be discovered is the Auxin and it was discovered by the biologist Charles Darwin. Auxins are one of the most important plant hormones. The chief naturally occurring auxin is indole-3 acetic acid – IAA   and other related compounds. Auxins are a group of phytohormones produced in the shoot and root apices and they migrate from the apex to the zone of elongation. The term, auxin was introduced by Kogl and Haagen- Smit (1931). Went (1928) isolated auxin from the Avena coleoptile tips by a method called Avena coleoptile or curvature test and concluded that no growth can occur without auxin. Auxins are widely distributed throughout the plant however, abundant in the growing tips such as coleoptile tip, buds, root tips and leaves. Indole Acetic Acid (IAA) is the only naturally occurring auxin in plants . The synthetic auxins include - IBA : Indole Butyric Acid, NAA : Naphthalene Acetic acid, MENA : Methyl ester of Naphthalene acetic acid, MCPA : 2 Methyl 4 chloro phenoxy acetic acid, TIBA : 2, 3, 5 Tri iodo benzoic acid, 2, 4-D : 2, 4 dichloro phenoxy acetic acid, 2, 4, 5-T : 2, 4, 5 – Trichloro phenoxy acetic acid.

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54. 6/12/2024 54 Developmental effects of Auxin Auxin regulates apical dominance.

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56. 6/12/2024 56 Developmental effects of Auxin Auxin delays the onset of leaf abscission. Auxin transported from the blade normally prevents abscission. Abscission is triggered during leaf senescence, when auxin is no longer being produced.

57. 6/12/2024 57 Developmental effects of Auxin Auxin promotes fruit development.

58. 6/12/2024 58 Auxin promotes the formation of lateral & adventitious roots. Cytokinin : promotes shoot growth in tissue culture -stimulates cell division in shoot apical meristem

59. 6/12/2024 59 Cytokinins Oppose apical dominance – promote outgrowth of lateral buds Regulate organogenesis in tissue culture Stimulate cell division in young shoots and leaves Delay leaf senescence Crown gall cells have acquired a gene for cytokinin synthesis

60. Gibberellins (Gas) GAs are known to be regulators of many phases of higher plant development, including seed germination, stem elongation, induction of flowering, pollen development and fruit growth. GA induces the synthesis of α -amylase in aleurone layer. Cereal grains can be divided into three parts: the embryo, the endosperm and the testa-pericarp . The endosperm is surrounded by aleurone layer. School of Biological Sciences and Biotechnology Haramaya University 60

61. Physiological Roles and Practical Applications of Abscisic acid (ABA) 1 . Inhibition of seed germination and seedling growth 2. Stomatal closing and resistance to stress 3 . Abscission and Senescence: 4 . Anti-gibberellin Effect: ABA generally inhibits the action of gibberellin in many plants called anti-gibberellin effect. It also inhibits GA promoted ER synthesis and β-amylase. 5 . Fruit Growth and Ripening : Practical application: In horticultural practices, for early ripening of fruits, ABA sprays will be helpful for premature harvest of fruits to yield more. Unripened Citrus and Tomato fruits due ABA treatment ripen early.

62. Transgenic Plants School of Biological Sciences and Biotechnology Haramaya University 62

63. School of Biological Sciences and Biotechnology Haramaya University 63 What are transgenic plants? ‘Transgenic’ indicates gene transfer using recombinant DNA technology. The transferred gene is usually, but not necessarily, from outside the normal range of sexual compatibility. Synonyms: Genetically modified organism (GMOs) Genetically engineered organism (GEOs) Living Modified Organism (LMOs ) Genetic Engineering and Recombinant DNA Technology : Genetic Engineering is the artificial manipulation, modification and recombination of DNA or other nuclei acid molecules from one organism into another organism.

64. School of Biological Sciences and Biotechnology Haramaya University 64 How to produce transgenic plants? (Techniques in plant Genetic engineering) Isolate and clone gene of interest Add DNA segments to initiate or enhance gene expression Add selectable markers Introduce gene construct into plant cells (transformation) Select transformed cells or tissues Regenerate whole plants

65. School of Biological Sciences and Biotechnology Haramaya University 65 Isolate and clone gene of interest The most limiting step in the transgenic process. Public and private labs are directing huge efforts to locate, identify, characterize, and clone genes of agricultural importance. This has led to a new era of ‘ Omics ’ .

66. Add DNA segments to control gene expression Promoter initiates transcription; affects when, where, and how much gene product is produced. Termination sequence marks end of gene. Promoter Termination sequence coding sequence

67. Transgene promoters Most commonly used is the CaMV 35S promoter of cauliflower mosaic virus. It is a constitutive promoter (turned on all the time in all tissues), and gives high levels of expression in plants. More specific promoters are under development: tissue-, time-, and condition-specific. Termination sequence Most commonly used is the nopaline synthase ( nos ) transcription terminator sequence from Agrobacterium tumefaciens .

68. School of Biological Sciences and Biotechnology Haramaya University 68 Add selectable markers Because gene transfer is an inefficient process (1 to 5% success rate), a system is needed to identify cells with the new genes. Typically, antibiotic or herbicide resistance genes are used as markers.

69. Antibiotic or herbicide resistance gene Promoter Gene of interest Termination sequence Expression cassette:

70. Introduce expression cassette into plant cells (transformation) “Gene gun” (biolistics, microprojectile bombardment) Agrobacterium infection

71. “Gene gun” method DNA Transformed plant cell plant chromosome inserted gene Gold particles DNA labeled Gold particles

72. Agrobacterium method disarmed T-DNA (contains transgene) gene transfer (Ti) plasmid Agrobacterium tumefaciens bacterial chromosome Transformed plant cell with gene plant chromosome inserted gene

73. Plant tissues used for transformation The choice of tissue depends on the species, but some common ones are immature embryos, leaf disks, and apical meristems. The tissue must be capable of generating callus (undifferentiated tissue), from which the complete plant can be produced. Various tissue culture independent methods are being developed and optimized.

74. To identify cells/tissues in which new genes are incorporated into plant’s DNA, grow in media containing antibiotics or herbicides. Successful transformant

75. Whole plants with inserted genes are regenerated through tissue culture.

76. Analyses of Transgenic plants DNA: PCR, Southern blot -- is the introduced DNA present in the plant’s genome? RNA: RT-PCR, Northern blot -- is mRNA produced? Protein: ELISA, Western blot -- is the protein produced? Bio-assays: Is the expected phenotypic trait observed?

77. Evaluate transformed plants: Presence and activity of introduced gene Other effects on plant growth Environmental effects Food or feed safety

78. Gene Transfer Vector-mediated gene transfer is carried out by a factor called Agrobacterium -mediate transformation Agrobacterium tumefaciens is a soil-borne gram-negative bacterium There are mainly two species of agrobacterium: A tumefaciens that induces Crown gall disease A rhizogene that induces hairy root disease Tumefaciens is a phytopathogen and is treated as the nature’s most effective plant genetic engineer 78 School of Biological Sciences and Biotechnology Haramaya University

79. Gene Transfer Cont’d 79 School of Biological Sciences and Biotechnology Haramaya University

80. Plasmid as Vectors Vector is an agent that carries gene from one organism to another Plasmids are self replicating circular DNA molecules in bacteria that are separate from and smaller than the bacteria chromosome, which carries gene in plant Vectors which carries gene in plants are: Tobacco mosaic virus Cauliflower mosaic virus 80 School of Biological Sciences and Biotechnology Haramaya University

81. Restriction Enzymes and DNA Ligase Restriction enzymes and DNA ligase are used to make recombinant DNA Restriction enzymes is a DNA cutting enzyme that recognizes specific site in DNA It produces two ends; sticky and blunt The most common restriction enzyme is the EcoRI DNA Ligase is a joining enzyme 81 School of Biological Sciences and Biotechnology Haramaya University

82. Restriction Enzymes and DNA Ligase Cont’d The steps involve; DNA fragment is firstly isolated Cutting of DNA Joining of DNA 82 School of Biological Sciences and Biotechnology Haramaya University

83. Cloning: Cloning is the process of genetically producing exact copy of a gene, cell or organism Cloning produces multiple copies of recombinant DNA A clone is an exact copy of a gene, cell or organism Recombinant DNA involves modifying pre-existing genes to form new genes T he most common plasmids used in cloning are: pbr322 and puc18 83 School of Biological Sciences and Biotechnology Haramaya University

84. Cloning Cont’d The are four steps involved in cloning: Isolation Ligation Transfection Selection 84 School of Biological Sciences and Biotechnology Haramaya University

85. Gene Cloning 85 School of Biological Sciences and Biotechnology Haramaya University

86. Polymerase Chain Reaction The polymerase chain reaction clones DNA without using cells. PCR takes place in a small tube that contains the sample DNA called primers. It includes all the four nucleotides of DNA coding (a, t, g and c) PCR is an indispensable tool Strength of PCR Weaknesses of PCR 86 School of Biological Sciences and Biotechnology Haramaya University

87. PCR 87 School of Biological Sciences and Biotechnology Haramaya University

88. Methods of transferring cloned gene into plant cells Biological (vector mediated) Agrobacterium Other bacteria Viruses Physical ( Nonvector ) Particle bombardment Electroporation Microinjection Sonoporation Laser induced Bead trnasfection

89. PHYSICAL Methods due to amphipathic nature of the phospholipid bilayer of the plasma membrane, polar molecules such as DNA and protein are unable to freely pass through the membrane. Various physical or mechanical methods are employed to overcome this and aid in gene transfer. 1. Electroporation 2. Microinjection 3. Particle Bombardment 4. Sonoporation 5. Laser induced 6. Bead transfection

90. Electroporation: Electroporation is a microbiological technique in which an electrical field is applied in cells in order to increase the probability of the cell membrane The basis of electroporation is the relatively weak hydrophobic/hydrophilic interaction of the phospholipids bilayer and ability to spontaneously reassemble after disturbance. A quick voltage shock may cause the temporary disruption of areas of the membrane and allow the passage of polar molecules. The membrane reseals leaving the cell intact soon afterwards. 90 School of Biological Sciences and Biotechnology Haramaya University

91. APPLICATIONS DNA transfection or transformation Electroporation is mainly used in DNA transfection/transformation which involves introduction of foreign DNA into the host cell (animal, bacterial or plant cell). Direct transfer of plasmids between cells It involves the incubation of bacterial cells containing a plasmid with another strain lacking plasmids but containing some other desirable features. The voltage of electroporation creates pores, allowing the transfer of plasmids from one cell to another. This type of transfer may also be performed between species. As a result, a large number of plasmids may be grown in rapidly dividing bacterial colonies and transferred to yeast cells by electroporation. Gene transfer to a wide range of tissues Electroporation can be performed in vivo for more efficient gene transfer in a wide range of tissues like skin, muscle, lung, kidney, liver, artery, brain, cornea etc. It avoids the vector-specific immune-responses that are achieved with recombinant viral vectors and thus are promising in clinical applications.

92. ELECTROPORATION Advantages It is highly versatile and effective for nearly all cell types and species.  It is highly efficient method as majority of cells take in the target DNA molecule. It can be performed at a small scale and onlya small amount of DNA is required as compared to other methods. Disadvantages Cell damage is one of the limitations of this method caused by irregular intensity pulses resulting in too large pores which fail to close after membrane discharge. Another limitation is the non-specific transport which may result in an ion imbalance causing improper cell function and cell death.

93. MICROINJECTION DNA microinjection was first proposed by Dr. Marshall A. Barber in the early of nineteenth century. This method is widely used for gene transfection in mammals.  It involves delivery of foreign DNA into a living cell (e.g. a cell, egg, oocyte, embryos of animals) through a fine glass micropipette. The introduced DNA may lead to the over or under expression of certain genes. It is used to identify the characteristic function of dominant genes.

94. PROCEDURE The delivery of foreign DNA is done under a powerful microscope using a glass micropipette tip of 0.5 mm diameter. Cells to be microinjected are placed in a container. A holding pipette is placed in the field of view of the microscope thatsucks and holds a target cell at the tip. The tip of micropipette is injected through the membrane of the cell to deliver the contents of the needle into the cytoplasm and then the empty needle is taken out.

95. ADVANTAGES No requirement of a marker gene. Introduction of the target gene directly into a single cell. Easy identification of transformed cells upon injection of dye along with the DNA. No requirement of selection of the transformed cells using antibiotic resistance or herbicide resistance markers. It can be used for creating transgenic organisms, particularly mammals.

96. Liposomes: Liposomes small spheres made of liquid molecule that can fuse easily with the plasma membrane. 96 School of Biological Sciences and Biotechnology Haramaya University

97. PARTICLE BOMBARDMENT Prof Sanford and colleagues at Cornell University (USA) developed the original bombardment concept in 1987 and coined the term “biolistics” (short for “biological ballistics”) for both the process and the device. Also termed as particle bombardment, particle gun, micro projectile bombardment and particle acceleration.  It employs high-velocity micro projectiles to deliver substances into cells and tissues.

98. USES This method is commonly employed for genetic transformation of plants and many organisms. This method is applicable for the plants having less regeneration capacity and those which fail to show sufficient response to  Agrobacterium - mediated gene transfer in rice, corn,wheat, chickpea,sorghum and pigeon-pea.

99. Particle Bombardment DNA- or RNA-coated gold/tungsten particles are loaded into the gun and you pull the trigger. A low pressure helium pulse delivers the coated gold/tungsten particles into virtually any target cell or tissue. The particles carry the DNA  cells do not have to be removed from tissue in order to transform the cells As the cells repair their injuries, they integrate their DNA into their genome, thus allowing for the host cell to transcribe and translate the transgene .

100. After shooting calli are placed on a selective media containing a herbicide for three weeks. Then calli are transferred to a media to induce the production of shoots . After they form small shoots, they are transferred to DARKER containers on a root induction media . plantsciences.montana.edu/ .../transform1.htm

101. The small plantlets are transplanted into soil and acclimated under high humidity conditions With current procedures only 10-20% of the plants are actually transgenic, so they should be tested on transgene expression

102. PARTICLE BOMBARDMENT ADVANTAGES Simple and convenient method involving coating DNA or RNA on to gold microcarrier , loading sample cartridges, pointing the nozzle and firing the device. No need to obtain protoplast as the intact cell wall can be penetrated. Manipulation of genome of sub-cellular organelles can be done. Eliminates the use of potentially harmful viruses or toxic chemical treatment as gene delivery vehicle. This device offers to place DNA or RNA exactly where it is needed into any organism DISADVANTAGES The transformation efficiency may be lower than  Agrobacterium- mediated transformation . Specialized equipment is needed. Moreover the device and consumables are costly. Associated cell damage can occur. The target tissue should have regeneration capacity. Random integration is also a concern. Chances of multiple copy insertions could cause gene silencing.

103. SONOPORATION Sonoporation involves the use of ultrasound for temporary permeabilization of the cell membrane allowing the uptake of DNA, drugs or other therapeutic compounds from the extracellular environment.  This method leaves the compound trapped inside the cell after ultrasound exposure.  It employs the acoustic cavitation of micro bubbles for enhancing the delivery of large molecules like DNA.The micro bubbles form complex with DNA followed by injection and ultrasound treatment to deliver DNA into the target cells.  Unlike other methods of transfection, sonoporation combines the capability to enhance gene and drug transfer.

104. SONOPORATION ADVANTAGES Simple and highly efficient gene transfer method. No significant damage is cause to the target tissue DISADVANTAGES Not suitable for tissues with open or cavitated structures. High exposure to low-frequency (<MHz) ultrasounds result in complete cellular death (rupture of the cell). Thus cellular viability must be taken into consideration while employing this technique.

105. LASER INDUCED INFECTION It involves the use of a brief pulse of focused laser beam.  In this method, DNA is mixed with the cells present in the culture and then a fine focus of laser beam is passed on the cell surface that forms a small pore sufficient for DNA uptake into the cells. The pore thus formed is transitory and repairs soon.

106. BEAD TRANSFECTION Bead transfection combines the principle of physically producing breaks in the cellular membrane using beads. In this method, the adherent cells are incubated for a brief period with glass beads in a solution containing the DNA. The efficiency of this rapid technique depends on: Concentration of DNA in a solution. Timing of the addition of DNA. Size and condition of the beads and the buffers utilized. Immunoporation is a recently developed transfection process involving the use of new type of beads, Immunofect TM  beads, which can be targeted to make holes in a specific type of cells.

107. Agrobacterium mediated gene transfer Mechanism (1) Sensing of plant chemical signals and inducing of virulence (vir) proteins . The chemical signals released by wounded plant are perceived by a VirA/VirG two-component system of A. tumefaciens , which leads to the transcription of virulence (vir) gene promoters and thus the expression of vir proteins. (2) T-DNA processing. T-DNA is nicked by VirD2/VirD1 from the T-region of Ti plasmid and forms a single-stranded linear T-strand with one VirD2 molecule covalently attached to the 5′end of the T-strand (3) Attaching of A. tumefaciens to plant and transferring of T-complex to plant cell . A. tumefaciens cell attaches to plant and transfers the T-complex from A. tumefaciens to plant cell by a VirD4/B T4SS transport system.

108. (4) Targeting of T-complex to plant cell nucleus and integrating of T-DNA into plant genome. The T-complex is transported into the nucleoplasm under the assistance of some host proteins and then integrated into plant genomic DNA. (5) Expressing of T-DNA in plant cell and inducing of plant tumor. The T-DNA genes encode phytohormone synthases that lead to the uncontrolled proliferation of plant cell and opine synthases that provide nutritive compounds to infecting bacteria.

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111. OMICS – Comprehensive analysis of the biological system . Large-scale biology – “OMICS” – Revolution in screening traits and develop novel improved organisms. term genomics was coined by Tom Roderick, a geneticist at the Jackson Laboratory (Bar Harbor , Maine), over beer at a meeting held in Maryland on the mapping of the human genome in 1986 The suffix - ome as used in molecular biology refers to a totality Similarly omics has come to refer generally to the study of large, comprehensive biological data sets OMICS 111

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113. D N A R N A Protein GENOMICS TRANSCRIPTOMICS PROTEOMICS Transcription Translation Lipids Nucleic acids Amino acids Sugars Phenotype METABOLOMICS PHENOMICS 113

114. Phenotypes produced by an organism Biomolecules found in an organism Proteins found in an organism mRNAs found in an organism Genes or genetic material in an organism S E T O F 114

115. GENOMICS Term by Thomas Roderick in 1986. Genomics : the branch of molecular biology concerned with the structure, function, evolution and mapping of genomes. Genome : the complete set of genes or genetic material present in a cell or organism. ( Winklen , 1920) The comprehensive study of whole sets of genes and their interactions •It investigates the variation in genes & how it affects protein structure and function throughout the life of a cell. 115 STEPS

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124. Genomics Functional genomics Functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects (such as genome sequencing projects) to describe gene (and protein) functions and interactions. Structural genomics Structural genomics seeks to describe the 3-dimensional structure of every protein encoded by a given genome. Epigenomics Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome Metagenomics Metagenomics is the study of metagenomes, genetic material recovered directly from environmental samples.

125. Genome Mapping Methods used to identify the locus of a gene and the distances between genes. Given by Alfred Sturtvent (1915) in Drosophila melanogaster. Traits mapped : Morphological Characters, Productivity traits, Resistance Traits, Quality Traits, Agronomic Traits, Special Characters. To study linkage and recombination. 125

126. Methods used for DNA sequencing Basic methods Sanger’s Method Maxam -Gilbert sequencing Chain-termination methods Large-scale sequencing and de novo sequencing Shotgun sequencing High-throughput methods Long-read sequencing methods Single molecule real time (SMRT) sequencing Nanopore DNA sequencing Short-read sequencing methods Massively parallel signature sequencing (MPSS) Polony sequencing 454 pyrosequencing Illumina ( Solexa ) sequencing Combinatorial probe anchor synthesis ( cPAS ) SOLiD sequencing Ion Torrent semiconductor sequencing DNA nanoball sequencing Heliscope single molecule sequencing Microfluidic Systems

127. Methods in development Tunnelling currents DNA sequencing Sequencing by hybridization Sequencing with mass spectrometry Microfluidic Sanger sequencing Microscopy-based techniques RNAP sequencing In vitro virus high-throughput sequencing

128. ‘ Make set of smaller clones from mapped ones Quality verification, Biological annotation, Submission to database Identify the set of genes in the region of genome Purify DNA from smaller clones. Perform sequencing chemistries Determine sequence from smaller clones Techniques to produce high quality sequences 128 Source : www.biotechonweb.com

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130. Some other techniques used in genomics Genomic DNA Isolation: As DNA is present in the nucleus of the cell, many methods are used to disrupt the external barriers like extracellular matrix, nuclear envelope and cell wall in case of plants. Separation of DNA: DNA molecules are negatively charged, so when they are subjected to an electric field they move from negative to positive electrode. For the movement of the DNA under the influence of electric current a medium is used which is called gel. This is a semisolid substance made from polymers like Agarose, Poly acrylamide etc . Cutting and Joining of DNA: The recombinant DNA technology mainly involves the precise cutting and joining of DNA molecules. Special types of enzymes are used to cut DNA at a particular site, these are called Restriction Enzymes.

131. Some other techniques used in genomics…. Cloning and Vectors: For inserting a piece of foreign DNA and to propagate the DNA fragment for large scale production a vector is used. Vectors are cloning vehicles which can replicate inside an appropriate host. Many different types of vectors are used for Molecular cloning—these are mainly Plasmid (circular double stranded DNA molecule), Cosmid (circular DNA molecules which can be also packaged inside a virus), bacteriophage (virus infecting different bacterial strains), etc. Detection of Gene of Interest: For detecting a particular gene of interest the techniques which are commonly used are Southern Hybridization, Colony Hybridization, Dot blot. The principle behind these methods is simple. The DNA molecule of our interest which we want to detect is called the Target. The DNA molecule with a known sequence with which we are going to detect the target molecule in a mixture of other DNA molecule is called a probe. Recombinant DNA and Cloning: Recombinant DNA is a chimeric DNA molecule which contains DNA from one source and some part of the DNA molecule is from another source. Production of Multiple Copies of DNA Using Polymerase Chain Reaction (PCR): Multiple copies of a DNA molecule can be produced by PCR. In 1983, biochemist Kary Mullis established the principle of polymerase chain reaction (PCR). This can only be done if parts of the sequence in question are known. School of Biological Sciences and Biotechnology Haramaya University 131

132. Genomics in medicine Diagnosis for example, where the cause of a range of symptoms cannot be pinpointed by any other means. Prenatal tests to screen, or where there is already a family history. It helps the parents to make informed choices and plans for the future. Where there is a family history of serious genetic disorders it can tell prospective parents whether or not they are a carrier and if they can pass it on to their children. if they are likely to develop the inherited condition later in life, even if they don't yet have any symptoms. To assess risk can show their susceptibility to suffer certain illnesses, like heart disease, stroke, and cancer. Possessing this knowledge means they can manage the risk through medicines, medical intervention, or making positive lifestyle changes.

133. Synthetic biology and bioengineering G enomic knowledge has enabled increasingly sophisticated applications of synthetic biology. Conservation genomics Conservationists can use the information gathered by genomic sequencing in order to better evaluate genetic factors key to species conservation, such as the genetic diversity of a population or whether an individual is heterozygous for a recessive inherited genetic disorder. By using genomic data to evaluate the effects of evolutionary processes and to detect patterns in variation throughout a given population . C onservationists can formulate plans to aid a given species without as many variables left unknown as those unaddressed by standard genetic approaches

134. COMPARATIVE GENOMICS WHAT IS COMPARATIVEGENOMICS? Analyzing & comparing genetic material from different species to study evolution, gene function, and inherited disease Understand the uniqueness between different species Comparative genomics is a powerful tool allowing us: To link genomic changes to environmental adaptation To transfer knowledge from model species to other plants To trace structural changes within a genome through time School of Biological Sciences and Biotechnology Haramaya University 134

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137. TRANSCRIPTOMICS Study of the Transcriptome. Transcriptome : complete set of RNA transcripts produced by the genome at any one time. Include mRNA, rRNA, tRNA, and other non-coding RNA. a .k.a Expression Profiling. To catalogue all species of transcripts. To determine the transcriptional structure of genes- start sites, 5′ and 3′ ends, splicing patterns and other PTMs. To quantify the changing expression levels of each transcript. Source : Various 137

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139. TRANSCRIPT PROFILING 139 Source : Genomic DNA Protein binding Crosslinking Protein Immunoprecipitation Protein digestion Labelling Microarray hybridization Antibody

140. 140 Applications and Scope 1 . Screening target genes 2. Predict gene function 3. Comparative transcriptomics helps in pattern of selection 4 . Role of comparative safety assessment of plant products ( GMO) 5. Identification of gene involving in stress 6. Understanding symbiotic association 7. Determination of pathogenicity function and Host pathogen interactions 8 . Dissection of food quality traits Expression of QTL isolation . Source : American Chemical Society, 2014

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149. PROTEOMICS Study about structure, function, composition and interaction of Proteome . Proteome : complete set of protein in a cell at a given time. By Mark Wilkins et.al in 1990’s. Helps in determining the proper treatment of diseases. Identification of B iomarkers . Pharmacoproteomics : The study of drugs using proteomics. 149

150. Proteomics I s the large-scale study of proteins The word proteome is a portmanteau of protein and genome was coined by Marc Wilkins in 1994. (While he was a Ph.D. student at Macquarie University.) The proteome is the entire set of proteins that is produced or modified by an organism or system. Proteomics is an interdisciplinary domain that has benefitted greatly from the genetic information of various genome projects, including the Human Genome Project. It covers the exploration of proteomes from the overall level of protein composition, structure, and activity. It is an important component of functional genomics.

151. M ore complicated than genomics because an organism's genome is more or less constant, whereas proteomes differ from cell to cell and from time to time. Distinct genes are expressed in different cell types, which means that even the basic set of proteins that are produced in a cell needs to be identified. Post-translational modifications T he translation from mRNA cause differences Many proteins also are subjected to a wide variety of chemical modifications after translation.

152. Interaction proteomics and protein networks the analysis of protein interactions from scales of binary interactions to proteome- or network-wide. Expression proteomics Expression proteomics includes the analysis of protein expression at larger scale Biomarkers “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention

153. Tools of Proteomics Peptide Mixture Protein Mixture Protein Peptides MS Analysis MS Data Identification separation separation digestion digestion d atabase search algorithms 153

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155. Using PAGE – Poly Acrylamide Gel Electrophoresis Softwares – BioNumerics2D, Delta2D , PDQuest , Progenesis Protein Topography and Migration Analysis Platform 155 Ionizes chemical species and sorts them into spectrum Based on their mass-to-charge ratio.

156. PURIFICATION SEQUENCE ANALYSIS QUANTIFICATION ANALYSIS CHARACTERIZATION STRUCTURAL ANALYSIS BIOINFORMATICS ANALYSIS PROCEDURE OF PROTEOMICS 156 Source : www.proteomics.com

157. Applications and Scope Arabidopsis - the role of GAs during initial stages of seed germination. Barley - cellular mechanisms under lying seed development during grain filling and seed maturation phases. Rice - novel traits useful for breeding. Maize - unknown novel genes coding for enzymes in metabolic pathways during grain development. Both abiotic and biotic stresses - manifested as the up- or down- regulation of proteins, or their post translation modification. Salinity stress - plant attempts to restore homeostasis in osmolarity to resume growth and development . P athogen attack - defence and stress related proteins, metabolic enzymes, translocation and protein turnover proteins. D ecipher the highly complex genetic interactions involved in plant-microbe interactions. For studying symbioses (nitrogen symbiosis, ecto - and endo-mycorrhizal symbiosis) in plants . Source : Rose et al., 2004 21

158. Methods of studying proteins Protein detection with antibodies (immunoassays) enzyme-linked immunosorbent assay (ELISA) western blot SDS-PAGE Antibody-free protein detection Detection methods Edman degradation, mass spectrometry-based techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Separation methods one-dimensional or two-dimensional separation

159. Hybrid technologies MSIA (mass spectrometric immunoassay) Current research methodologies Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE) High-throughput proteomic technologies Mass spectrometry and protein profiling Protein chips Reverse-phased protein microarrays

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161. Applications of Proteomics Interaction proteomics and protein networks Most proteins function via protein–protein interactions, and one goal of interaction proteomics is to identify binary protein interactions, protein complexes, and interactomes . Expression proteomics It helps identify main proteins in a particular sample, and those proteins differentially expressed in related samples—such as diseased vs. healthy tissue Biomarkers Understanding the proteome, the structure and function of each protein and the complexities of protein–protein interactions is critical for developing the most effective diagnostic techniques and disease treatments in the future. Proteogenomics Parallel analysis of the genome and the proteome facilitates discovery of post-translational modifications and proteolytic events, especially when comparing multiple species Structural proteomics includes the analysis of protein structures at large-scale. It compares protein structures and helps identify functions of newly discovered genes.

162. Bioinformatics for proteomics Protein identification there are currently programs available for protein identification. These programs take the peptide sequences output from mass spectrometry and microarray and return information about matching or similar proteins. Protein structure through bioinformatics, there are computer programs that can in some cases predict and model the structure of proteins. These programs use the chemical properties of amino acids and structural properties of known proteins to predict the 3D model of sample proteins. This also allows scientists to model protein interactions on a larger scale

163. Post-translational modifications computational analysis of post-translational modifications The current post-translational modification programs are only predictive. Chemists, biologists and computer scientists are working together to create and introduce new pipelines that allow for analysis of post-translational modifications for their effect on the protein's structure and function. Computational methods in studying protein biomarkers One example of the use of bioinformatics and the use of computational methods is the study of protein biomarkers. Computational predictive models have shown that extensive and diverse feto -maternal protein trafficking occurs during pregnancy and can be readily detected non-invasively in maternal whole blood.

164. Emerging trends in proteomics Proteomics for systems biology Advances in quantitative proteomics would clearly enable more in-depth analysis of cellular systems. Transcriptional and translational responses to perturbations (cell cycle, cellular differentiation, carcinogenesis, environment (biophysical), etc.) results in functional changes to the proteome implicated in response to the stimulus Human plasma proteome Biological systems are subject to a variety of perturbations (cell cycle, cellular differentiation, carcinogenesis, environment (biophysical), etc.). Transcriptional and translational responses to these perturbations results in functional changes to the proteome implicated in response to the stimulus. It also contains tissue leakage proteins due to the blood circulation through different tissues in the body.

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166. METABOLOMICS Study of M etabolome . Metabolome : collection of all metabolites in a cell, tissue, organ or organism . Metabolites are ultimate result of cellular pathways. Look at genotype- phenotype as well as genotype- environ type relationships. Metabolic profiling : Quantitative study of a group of metabolites, known or unknown, within or associated with a particular metabolic pathway. Metabolic fingerprinting : Measures a subset of the whole profile with little differentiation or quantitation of metabolites. Monitoring crop quality characteristics Source : Various 167

167. Metabolomics Metabolomics is the scientific study of chemical processes involving metabolites, the small molecule substrates, intermediates and products of metabolism. Specifically , metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind", the study of their small-molecule metabolite profiles. Metabonomics Defined as "the quantitative measurement of the dynamic multi-parametric metabolic response of living systems to pathophysiological stimuli or genetic modification" . The word origin is from the Greek μετ αβολή meaning change and nomos meaning a rule set or set of laws.

168. Metabolome Metabolome refers to the complete set of small-molecule (<1.5 kDa ) metabolites (such as metabolic intermediates, hormones and other signalling molecules, and secondary metabolites) to be found within a biological sample, such as a single organism. The metabolome is dynamic, changing from second to second . Each type of cell and tissue has a unique metabolic ‘fingerprint’ that can elucidate organ or tissue-specific information. Bio-specimens used for metabolomics analysis include but not limit to plasma, serum, urine, saliva, faeces, muscle, sweat, exhaled breath and gastrointestinal fluid It is not currently possible to analyse the entire range of metabolites by a single analytical method

169. 1. Metabolite Extraction 2. Chromatography 3. Mass Spectrometry 4. Data Analysis [ Rapid Enzyme Quenching ] [ Separation ] [ Specificity ] [ Metabolite identification ] Cell & Tissues Leaves & other organs Cold organic solvents Grinding Weighing Samples 170 Source : www.metabolon.com

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171. Applications and scope of metabolomics Identifying potential biochemical markers optimize trait development in agricultural products and in bio refining. Differentiate genotypes and phenotypes based on metabolic levels. Differentiating various genotypes and understanding plant responses to biotic and abiotic stresses . C haracterization of the novel plant products . Comparison between transgenic and wild-type plants. I mproved levels of phytonutrients such as flavonoids and carotenoids . Plant properties are improved - increasing metabolic fluxes into valuable biochemical pathways using metabolic engineering. e.g., nutritional value of foods, decreasing the need for pesticide or fertilizer application etc. 8. Into pathways needed for the production of pharmaceuticals in plants. Introducing foreign set of enzymes that lead to the production of desired end products and new metabolites. Sources : Wishart , 2007 172

172. Key applications Toxicity assessment/toxicology by metabolic profiling detects the physiological changes caused by toxic insult of a chemical For functional genomics excellent tool for determining the phenotype caused by a genetic manipulation, such as gene deletion or insertion Metabologenomics a novel approach to integrate metabolomics and genomics data by correlating microbial-exported metabolites with predicted biosynthetic genes Fluxomics Is a further development of metabolomics . The disadvantage of metabolomics is that it only provides the user with steady-state level information, while fluxomics determines the reaction rates of metabolic reactions and can trace metabolites in a biological system over time. Nutrigenomics term which links genomics, transcriptomics , proteomics and metabolomics to human nutrition.

173. Genome can tell what could happen, T ranscriptome can tell what appears to be happening , Proteome can tell what makes it happen and Metabolome can tell what has happened and what is happening.

174. PHENOMICS Study of the Phenome. Phenome : sum total of all phenotypes produced by an organism. P henotypes are characterized in a rigorous and formal way, and are linked to the associated genes and gene variants (alleles ). Genotype –Phenotype map is made to analyse an organism. 175 Source : www.frontiersin.org

175. PHENOMICS ADVANTAGES Identify relation between Genotypes and Phenotypes. Assess pleotropic effects. Assess phenotypic quality, Study relation of phenotypes with environment. Study characters like: DIS ADVANTAGES Time consuming. High chance of mistake. Effect of environment is very crucial. Mutation cannot be taken into account. 176 Source : http://www.phenomecentre.org Plant height, Leaf area, Chlorophyll content & Photosynthetic efficiency, Necrosis, Growth rate, Canopy temperature, Ear/panicle size/number, Salinity/drought/heat /frost tolerance, Root mass/growth, Biomass, Transpiration rate etc.

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179. Applications of Plant Biotech. 180 School of Biological Sciences and Biotechnology Haramaya University

180. Schematic presentation of applications of plant biotechnology in agriculture Storage of genetic resources via Biodiversity and gene flow, Species under threat, ----Investment of Iraqi plant wealth for new pharmaceuticals 181 School of Biological Sciences and Biotechnology Haramaya University

181. Role of plant biotechnology in plant based industry Steroids , food additives, insecticides 182 School of Biological Sciences and Biotechnology Haramaya University

182. Production of Pharmaceuticals Large scale, up to 10.000 liter bioreactors, No seasonality, specific target compounds, medicine, food preservatives, pigments, flavors, fragrance, essential oil Increase drugs of natural sources increase in income. Food security. 183 School of Biological Sciences and Biotechnology Haramaya University

183. Production of Therapeutic antibodies in plants In 1990, the first therapeutic protein expressed in plants was human serum albumin produced in tobacco and tomato leaves. Since then: over 100 pharmaceutical proteins have been produced in plants. i.e. Plants are drug factories 184 School of Biological Sciences and Biotechnology Haramaya University

184. Edible Vaccines vis Molecular farming, Gene(s) induce immunity can be expressed in edible food Strawberry or apple rather than injection 185 School of Biological Sciences and Biotechnology Haramaya University

185. Environmental Biotechnology Phytoremediation ? 186 School of Biological Sciences and Biotechnology Haramaya University

186. Phytoremediation Plants for cleaning up the environment Removal of contaminants from soil, water, air, e.g. heavy metals, CO, CO 2 , TNT, oil splits …etc Healthy environment. 187 School of Biological Sciences and Biotechnology Haramaya University

187. Conventional Remediation 188 School of Biological Sciences and Biotechnology Haramaya University

188. Gene Banks vis Storage of Genetic Resources Plant tissues can be Preserved for millions of years, thereafter can be regenerated. Imagine the differences between gene banks & seed banks!!!!!!! Millions of $$$$ can be saved 189 School of Biological Sciences and Biotechnology Haramaya University

189. Biofuel vis Biogas # Landfills Methane gas # One kg of sunflower 37 megajoules of energy # One kg of crude oil 42 megajoules of energy In Brazil, sugar cane Ethanol In U.S.A., sweet potato is a candidate for future Biofuel In U.S.A., 10% of highway vehicles use soya bean oil 8 kg 1.5 gallon of Biofuel 190 School of Biological Sciences and Biotechnology Haramaya University

190. Algal farming for Biofuel 191 School of Biological Sciences and Biotechnology Haramaya University

191. Fuel + Ornament 192 School of Biological Sciences and Biotechnology Haramaya University

192. Plant Biotechnology A crown gall is the result of an infection by a common soil bacterium, Agrobacterium tumifaciens. The unique properties of this bacterium have made it a useful tool in genetic engineering of plants. 193 School of Biological Sciences and Biotechnology Haramaya University

193. Next Green Revolution Inoculation of non Legumes with Nitrogen fixation bacteria nif genes Huge reduction in Nitrogen fertilizers Approx. a reduction in farming cost by 20%. 194 School of Biological Sciences and Biotechnology Haramaya University

194. Strategies of gene transfer in plants 195 School of Biological Sciences and Biotechnology Haramaya University

195. Genetic Engineering Transfer of desired gene(s) to plants There are more than ten methods Produced Hundreds of new varieties Easiest and common is gene gun 196 School of Biological Sciences and Biotechnology Haramaya University

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197. Wool genes 198 School of Biological Sciences and Biotechnology Haramaya University

198. Manipulation of Chloroplasts (RUBISCO enzymes) More Photosynthesis Storage of more food. 199 School of Biological Sciences and Biotechnology Haramaya University

199. Manipulation of Ion Pumping & Transportation Absorption of more ions Better plant growth more yield 200 School of Biological Sciences and Biotechnology Haramaya University

200. Quality improvement Improvement of nutritional quality of crops, e.g. primary metabolites, & therapeutic e.g. secondary metabolites. Wheat, barley, maize are low in lysine. Legumes are deficient in S containing amino acids Therefore Breed for such traits 201 School of Biological Sciences and Biotechnology Haramaya University

201. Genetic Engineering Genetic engineering transfers genes from varieties of organisms into plants. Genetic engineering has made pest resistant plants This engineering has also made plants more productive As well they are resistant to pests 202 School of Biological Sciences and Biotechnology Haramaya University

202. Genetic Engineering Cont’d Plants has been engineered to resist pests viruses and fungi This resistance occurs because the plants produce the proteins which prevent the virus from attaching to plant cell It also blocks the replication of genetic codes in plants This production of proteins helps plants like papaya against devastating spot ring virus 203 School of Biological Sciences and Biotechnology Haramaya University

203. Transgenic Plants Benefits of transgenic plants to humans Medical benefits Industrial benefits Agricultural benefits Transgenic plants improves poor access of food 204 School of Biological Sciences and Biotechnology Haramaya University

204. Genetically Engineered Crops Require Extensive Field and Market Testing Before They Are Released Field testing is creating, using and iterating your offering before offering it to customers Test marketing is an experiment conducted in a field laboratory (the test market) comprising of actual stores and real life buying situations ,without the buyers knowing they are participating in an evaluation exercise The sensitivity of a new plant may have increased in fungal disease. Time frame is 6yrs before it is available on the markets 205 School of Biological Sciences and Biotechnology Haramaya University

205. Genetically Engineered Plants Must Be Safe For Environment and Consumers It helps in the reduction of malnutrition It provides vitamins such as vitamin a, iron, zinc, protein, essential amino acids and essential fatty acids It reduces the level of anti nutritional factors such as cyanogens etc 206 School of Biological Sciences and Biotechnology Haramaya University

206. Advantages of Genetically Modified Crops Nutritional content can be improved Genetically modified foods can have a longer shelf life We receive medical benefits from GM crops It creates foods that are more appealing to eat GM foods are easier to transport 207 School of Biological Sciences and Biotechnology Haramaya University

207. Disadvantages of Genetically Modified Crops GM crops may cause antibiotic resistance Genes go into different plant species Independent research is not allowed 208 School of Biological Sciences and Biotechnology Haramaya University

208. Brief History On The First Genetically Modified Crop The first genetically modified food approved for release was the Flavr Savr tomato in 1994. It was developed by a company called Calgene and it was engineered to have a longer shelf life by inserting an antisense gene that delayed ripening. 209 School of Biological Sciences and Biotechnology Haramaya University

209. Golden Rice Golden rice is a variety of rice ( oryza rice) produced through genetic engineering to biosynthsize beta-carotene, a precursor in the edible parts of rice. It is intended to produce a fortified food to be grown and consumed in areas with a shortage of dietary vitamin a, a deficiency which each year is estimated to kill 670,000 children under the age of five (5) and cause an additional 500,000 causes of irreversible childhood blindness. It was done in the philippines in 1999. 210 School of Biological Sciences and Biotechnology Haramaya University

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