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Micropropagation and transformation
This document summarizes micropropagation techniques used to rapidly multiply plants in vitro. It discusses explant selection, initiation and establishment of aseptic cultures, multiplication of plantlets, and acclimatization. Micropropagation allows for mass production of disease-free plants through small propagules in controlled environments. The document also covers plant genetic transformation methods using Agrobacterium tumefaciens to introduce exogenous DNA and produce transgenic plants with desired traits.
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Micropropagation and transformation
- 1. Micropropagation and transformation principles, uses and methods
- 2. By M. Sc.Student Mohamed Salaheldin Mokhtar Under the supervision of Prof. Dr. Mohamed Abdelbaaeth Elseehy
- 5. the art andscience of multiplying plants in vitro
- 6. Rapid clonal invitro propagation of plants from cells, tissues or organs cultured aseptically on defined media contained in culture vessels maintained under controlled conditions of light and temperature …
- 7. Explant Cell, tissueor organ of a plant that is used to start in vitro cultures.
- 8. totipotency The capacityof a cell (or a group of cells) to give rise to an entire organism.
- 9. multiply novel plants Micropropagation is used to provide a sufficient number of plantlets for planting from a stock plant which… does not produce seeds does not respond well to vegetative reproduction
- 10. MICRO. Vs MACRO. PROPAGATION • Small propagule • Aseptic conditions • Controlled environment • Heterotrophic growth • Rapid multiplication • Greater initial costs • Larger propagule • Non-aseptic conditions • Less environmental control • Photoautotrophic growth • Slower multiplication • Nominal costs
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- 12. Establishment Selection of explant Initiation and aseptic culture establishment
- 14. Selection of explant • Explant age • Season • Explant size • Plant quality • Goal
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- 16. In many cases,older tissue will not form callus younger tissue easier to surface disinfect
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- 20. EXPLANT SIZE •the smaller the explant, the harder it is to culture. • The larger explants probably contain more nutrient reserves and plant growth regulators to sustain the culture.
- 21. Internal differences inhormone balance in the tissue can result in varying in vitro responses.
- 22. PLANT QUALITY •It is advisable to obtain explants from plants which are healthy as compared to plants under nutritional or water stress or plants which are exhibiting disease symptoms.
- 23. GOAL • Dependingon what type of a response is desired from the cell culture, the choice of explant tissue will vary.
- 24. • Any pieceof plant tissue can be used as an explant
- 25. • For example,if clonal propagation is the goal, then the explant will usually be a lateral or terminal bud or shoot. • For callus induction, pieces of the cotyledon, hypocotyl, stem, leaf, or embryo are usually used. • Excellent explants for callus induction are seedling tissues from aseptically germinated seeds or immature inflorescences. • Leaf tissue from the aseptically germinated seed is a good source of tissue for protoplast isolation. • To produce haploid plants or callus, the anther or pollen is cultured.
- 27. Initiation and asepticculture establishment • The explant is surface sterilized before being placed on the medium. Small amounts of plant growth regulators may be added to the medium for quick establishment of the explant.
- 28. Aseptic Technique •Killing or excluding microorganisms or their spores with heat, filters, chemicals or other sterilants
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- 36. • Liquid laundrybleach (NaOCl at 5-6% by vol) 1.Rinse thoroughly after treatment 2.Usually diluted 5-20% v/v in water; 10% is most common • Calcium hypochlorite – Ca(OCl)2 • a powder; must be mixed up fresh each time • Ethanol (EtOH) 1.95% used for disinfesting plant tissues 2.Kills by dehydration 3.Usually used at short time intervals (10 sec – 1 min) 4.70% used to disinfest work surfaces, worker hands • Isopropyl alcohol (rubbing alcohol) is sometimes recommended
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- 38. Macronutrients Media Micronutrients Carbon and energy sources Vitamins and myo-inositol Growth regulators Solidifying agents
- 39. Macronutrients 1. Carbon(C) 2. Nitrogen (N) 3. Potassium (K) 4. Hydrogen (H) 5. Calcium (Ca) 6. Phosphorus (P) 7. Oxygen (O) 8. Magnesium (Mg) 9. Sulphur (S)
- 40. Micronutrients 1. Iron(Fe) 2. Sodium (Na) 3. Chlorine (Cl) 4. Manganese (Mn) 5. Zinc (Zn) 6. Boron (B) 7. Copper (Cu) 8. Molybdenum (Mo) 9. Nickel (Ni)
- 41. Carbon and energysources
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- 44. Growth regulators AuxinsCytokinins Gibberellins abscisic acid Ethylene Other
- 46. Auxin and CytokininRatio
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- 48. Multiplication • isthe taking of tissue samples produced during the first stage and increasing their number
- 52. Pretransplant Rooting hardening • treating the plantlets/shoots produced to encourage root growth and "hardening." It is performed in vitro, or in a sterile "test tube" environment
- 54. Transfer from cultureto the natural environment or acclimatization • the plantlets are removed from the plant media and transferred to soil or (more commonly) potting compost for continued growth by conventional methods.
- 58. Advantages Rapid &efficient propagation Year-round production Reduce stock plant space Long-term germplasm storage Production of difficult-to-propagate species Production of disease-free plants
- 59. Disadvantages X Equipment/facilityintensive operation X Technical expertise in management positions X Protocols not optimized for all species X Liners may not fit industry standard X It may be too expensive
- 60. Seed culture Typesof culture (Explant base) Embryo culture Cell culture (suspension culture) Callus Bud culture culture Meristem culture Protoplast culture Organ culture
- 61. Applications of TissueCulture 1. Embryo culture 2. Meristem culture 3. Micropropagation 4. Somatic embryogenesis and Organogenesis 5. Somaclonal variation and in vitro selection 6. Anther culture Haploid & Dihaploid Production 7. Protoplast culture (In vitro hybridization – Protoplast Fusion) 8. Germplasm preservation
- 64. Plant Genetics Transformation • transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
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- 68. Direct biolistics or particle bombardment electroporation Polyethyleneglycol Microinjection
- 69. Biolistics or particle bombardment
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- 75. Crown Gall on Tobacco Infection of a plant with A. tumefaciens and formation of crown galls
- 78. Gene transfer inplants Why gene transfer? • Crop improvement • Disease resistance • Stress tolerance • Improved performance • Value-added traits Basic studies • Gene expression • Reverse genetics - understanding functioning of unknown genes • Biochemistry and metabolism
- 79. Plant transformation withthe Ti plasmid of Agrobacterium tumefaciens • A. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in crown gall (cancer) tumors • Tumor formation is the result of the transfer, integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA (transferred DNA) • The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found in A.tumefaciens
- 80. Overview of requirementsfor plant genetic transformation • Trait that is encoded by a single gene • A means of driving expression of the gene in plant cells (Promoters and terminators) • Means of putting the gene into a cell (Vector) • A means of selecting for transformants • Means of getting a whole plant back from the single transformed cell (Regeneration)
- 82. Transformation with Agrobacterium • Agrobacterium Bacterial contains a circle of DNA (Ti plasmid) that carries the desired genes • Co-cultivation of the Agrobacterium with plant pieces transfers the DNA Ti Plasmid chromosome Petri dish with leaf pieces plus Agrobacterium
- 84. Essential Elements forCarrying a Transgene on Ti Plasmids The T-DNA segment contains both a transgene and a selective marker or reporter gene. These have separate promoters and termination signals. The marker or reporter gene must be expressed all the time, whereas the transgene is often expressed only in certain tissues or under certain circumstances and usually has a promoter that can be induced by appropriate signals.
- 85. • Selected singlecells from the callus can be treated with a series of plant hormones, such as auxins and gibberellins, and each may divide and differentiate into the organized, specialised, tissue cells of an entire plant. The new plant that originated from a successfully shot cell have new genetic (heritable) traits.
- 86. Transfer of ModifiedTi Plasmid into a Plant Agrobacterium carrying a Ti plasmid is added to plant tissue growing in culture. The T-DNA carries an antibiotic resistance gene (neomycin in this figure) to allow selection of successfully transformed plant cells. Both callus cultures (A) and liquid cultures (B) may be used in this procedure.
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- 89. Development of GMfoods 1950 First regeneration of entire plants from an in vitro culture 1973 Researchers develop the ability to isolate genes 1983 1st transgenic plant: antibiotic resistant tobacco 1990 First successful field trial of GM cotton- CROP Flavr-Savr tomato - 1st FDA approval for a food 1995 Monsanto's Roundup Ready soybeans approved for sale in the United States. 1994 GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS 1985