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Plant Tissue Culture, Methods and Applications
The document discusses plant tissue culture methods, including definitions, history, and applications in agriculture and biotechnology. Key concepts such as plasticity and totipotency are highlighted, along with stages of micropropagation and factors affecting tissue culture success. It emphasizes the importance of tissue culture in generating disease-free plants and improving food security through advancements in various fields.
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Plant Tissue Culture, Methods and Applications
- 1. PLANT TISSUE CULTURE METHODS &APPLICATIONS By OJUEDERIE Omena Bernard Department of Biotechnology College of Food Sciences Bells University of Technology Ota.
- 2. Introduction Tissue culture “… Amethod of biological research in which fragments of tissues from an animal or plant are grown in vitro in artificial medium under aseptic conditions and continue to survive and function.”
- 3. Introduction What is planttissue culture? “… the aseptic culture of plant protoplasts, cells, tissues or organs under aseptic conditions which lead to cell multiplication or regeneration of organs or whole plants.”
- 4. Basic concepts ofplant tissue culture(PTC)Two concepts, are central to understanding plant cell, tissue, organ culture and regeneration. Plasticity. -ability to initiate cell division from almost any tissue of the plant. -ability to regenerate lost organs or undergo developmental pathways in response to particular stimuli. Totipotency. -each cell has the capacity to regenerate the entire plant.
- 5. Basic concepts ofplant tissue culture(PTC) Cells lines differentiate to form specialized tissues and organs. Unlike animal cells, living plant cells re-differentiate. Therefore, tissue can be regenerated from explants such as cotyledons, hypocotyls, leaf, ovary, protoplast, petiole, root, anthers, etc.
- 6. Short history ofPlant Tissue Culture A German plant physiologist Gottlieb Haberlandt (1902) cultured isolated single palisade cells from leaves in Knop's salt solution enriched with sucrose. Haberlandt is regarded as the father of plant tissue culture.
- 7. Short history ofPlant Tissue Culture Toward Commercial Micropropagation 1950s Morel & Martin 1952 used Meristem-tip culture for disease elimination. They recovered for the first time, virus- free Dahlia plants
- 8. Short history ofPlant Tissue Culture Morel 1960 Disease eradication Wimber 1963 & in vitro production of orchids
- 9. Short history ofPlant Tissue Culture Commercialization of Micropropagation Murashige and Skoog(1962) Developed a universally used high salt medium containing mineral salts, vitamins, an energy source and growth hormone (MS medium) Murashige(1974) Broad commercial application
- 10. What is required? Tissueculture has several critical requirements: Appropriate tissue. A suitable growth medium which can be liquid or semisolid. Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture.
- 12. Tissue culture environment General Laboratoryand media preparation room Transfer area Culturing facilities/Growth rooms Washing and storage rooms Greenhouses and containment room
- 13. General laboratory &media
- 14. Transfer area (GRCIITA IBADAN)
- 15. Design of aLaminar flow hood
- 16. A section ofa culture room Timer switch to control photoperiod Fluorescent tube placed above shelf for lighting A level of shelf containing in vitro cultures
- 17.
- 18. Micropropagation “… the artand science of multiplying plants in vitro It implies - regeneration - multiplication - uniformity ??
- 19. Starting material for Micropropagation (Explant) Propagationfrom plant cells, tissues or organs under aseptic conditions in synthetic medium in vitro. Many different explants can be used for micropropagation, but axillary buds and meristems are most commonly used Tip bud Leaf Axillary bud Internode Root
- 20. Stages of Micropropagation Stage I- Selection & preparation of the mother plant – sterilization of the plant tissue takes place 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
- 21. Stages of micropropagation STAGEIV: Transfer to Natural Environment STAGE III: Pretransplant (rooting) STAGE II: Shoot Production STAGE I - Sterilization
- 22. Growth medium The growthmedium used depends on the plant species being grown. The medium contains the following components: all of the minerals and vitamins needed for plant growth and differentiation; a carbon/energy source such as sugar as the explant cannot usually photosynthesize; various growth regulators to encourage cell enlargement, division and differentiation; agar which is used to solidify the medium.
- 23. Plant growth regulators Twomajor hormones affect Plant Differentiation:– Auxins: Stimulates Root Development & Cytokinin: Stimulates Shoot Development Generally, the ratio of these two hormones can determine plant development: – ↑ Auxin ↓Cytokinin = Root Development – ↑ Cytokinin ↓Auxin = Shoot Development – Auxin = Cytokinin = Callus
- 24. Hormonal balance Auxin Cytokinin HighLow Root formation on cuttings Embryogenesis Adventitious root formation in callus Callus initiation Adventitious shoot formation Axillary shoot growth Low High
- 25. Methods of micropropagation Clonal propagationinvolves the multiplication of genetically identical lines by asexual reproduction. Micropropagation involves the use of bud culture, meristem and shoot-tip culture techniques to introduce plants in vitro by induction to form adventitious buds, shoots and entire plants.
- 26. Methods of micropropagation cont’d Bud cultureis the culture of plant buds which contains active meristems. Meristem culture is the culture of apical meristems which are capable of active cell division and differentiation into specialized and permanent tissue such as shoots and roots
- 27. Methods of micropropagation cont’d Single nodeculture is done on a hormone free medium. Axillary bud culture is done using excised shoot tips cultured on medium amended with high cytokinin concentration 6 Benzyl amino purine(BAP)/Benzyl adenine(BA)
- 28. Bud culture bysingle nodes Surface sterilization in NaOCL Rinsing with SDW Transfer into sterile container for 2nd sterilization Steps in single node culture 1 2 3 4 5
- 29. Methods of micropropagation cont’d Micro cuttingsof yam during and after subculture 3 4
- 30. In vitro plantlet regenerationvia meristem & shoot tip culture
- 31. Meristem culture A meristemis an undifferentiated but determined tissue, the cells of which are capable of active cell division and differentiation into specialized and permanent tissues such as shoots and roots. It has the capacity of producing a complete plant in vivo and in vitro (Totipotent)
- 32.
- 33. Meristem excison usingstereomicroscopes
- 34. Dumet et al.,2008
- 35. Shoot tip culture Clonalin vitro propagation by repeated enhanced formation of axillary shoots from shoot-tips cultured on media supplemented with plant growth regulators, usually cytokinins. The size of the shoot tip is between 0.5 to 5mm and consists of primordia leaves.
- 37. Stages in acclimatizationof Cassava 1 2 3 4 5
- 38. Acclimatization of Yamusing constructed humidity chamber Acclimatization process using polyethylene
- 40. Problems in Micropropagation There isproblem of genetic variability due to Somaclonal variation in some cultures. Contamination is a major problem which could cause high losses within a short period. It requires expensive equipments and well trained manpower. Vitrification may occur which reduces the rate of growth multiplication of the plant and eventually causes death.
- 41. Benefits of micropropagation Rapid multiplicationof superior clones can be carried out through out the year, irrespective of seasonal variations. Multiplication of disease free plants e.g. virus free plants of sweet potato (Ipomea batatas), cassava (Manihot esculenta) e.t.c. It is a cost effective process as it requires minimum growing space.
- 42. Growth Media– Minerals,Growth factors, Carbon source, Hormones Environmental Factors– Light, Temperature, Photoperiod. Explant Source– Usually, the younger, less differentiated the explant, the better for tissue culture Genetics– Different species show differences in amenability to tissue culture. In many cases, different genotypes within a species will have variable responses to tissue culture. Factors affecting Tissue Culture
- 43. Applications of plant tissueculture Agriculture Anther & pollen culture-Production of haploid plants.
- 44. Applications of plant tissueculture Production of virus-free plants for safe germplasm transfer. Screening of in vitro lines for stress and disease resistance. Somaclonal Variations has been used in plant breeding programmes where the genetic variations with desired or improved characters are introduced into the plants.
- 45. Somaclonal variation Somaclonal variationis a general phenomenon of all plant regeneration systems that involve a callus phase. There are two general types of Somaclonal Variation: Heritable, genetic changes (alter the DNA). Stable, but non-heritable changes (alter gene expression).
- 46. Germplasm conservation An extensionof micropropagation techniques through two methods: Slow growth techniques e.g.: ↓ Temp., ↓ Light, media supplements ( growth retardants).Medium-term storage (1 to 4 years) Cryopreservation.Ultra low temperatures in liquid nitrogen at -196o C. Stops cell division & metabolic processes Very long-term (indefinite?)
- 47. Clonal germplasm flowfrom medium- term storage to long term storage D.Dumet, 2009. IITA
- 48. Cryopreservation Micropropagation Unit, RoyalBotanic Gardens, Kew UK. Cryopreservation is a valuable technology for the cost- effective,sustained long-term conservation of plant genetic material.
- 49. Cryopreservation Requirements Preculturing– Usually arapid growth rate to create cells with small vacuoles and low water content. Cryoprotection– Glycerol, DMSO, PEG, etc…, to protect against ice damage and alter the form of ice crystals. Freezing– The most critical phase; one of two methods is used: Slow freezing which allows for cytoplasmic dehydration. Quick freezing which results in fast intercellular freezing with little dehydration.
- 50. Cryopreservation Requirements Storage – Usuallyin liquid nitrogen (-196o C) to avoid changes in ice crystals that occur above -100o C Thawing– Usually rapid thawing to avoid damage from ice crystal growth. Recovery (don’t forget you have to get a plant) – Thawed cells must be washed of cryoprotectants and nursed back to normal growth. – Avoid callus production to maintain genetic stability.
- 51. Applications of plant tissueculture Horticulture and Forestry: Micropropagation method is used for rapid multiplication of ornamental plants as well as important trees yielding high fuel, pulp, fruits or oil at a large scale.
- 52. Applications of plant tissueculture Today's high yielding oil palms on plantations in Malaysia were generated in the 1960s by tissue culture methods. These palms produce 30% more oil than normally cultured palms. Improvement of economically important forest trees is being done through genetic transformation and rapid Micropropagation .e.g in vitro regeneration and genetic transformation of conifers.
- 53. Applications of plant tissueculture Industries: Plant cell culture is used for biotransformation (modification of functional groups of organic compounds by living cells). Food and agricultural biotechnologists are involved in using tools of molecular biology to enhance the quality and quantity of foods and economic crops. For example, Golden Rice was genetically enhanced with added beta carotene, which is a precursor to Vitamin A in the human body. Plant cells can be cultured in fermenters for the industrial production of secondary Metabolites using cell culture.
- 54. PLANT SPECIES AND SECONDARYMETABOLITES OBTAINED FROM THEM USING TISSUE CULTURE TECHNIQUES Product Plant source Uses Artemisin Artemisia spp Antimalarial Capsaicin Capsicum annum Cures Rheumatic pain Codeine Papaver spp. Analgesic Camptothecin Campatotheca accuminata Anticancer Quinine Cinchona officinalis Antimalarial
- 55. At Bells University Planttissue culture practicals Capsicum annum (Red Pepper) regenerated via seed culture Apple regenerated via embryo culture Apple subcultured into test tubes using
- 56.
- 57. Possible areas forresearch Somatic embryogenesis of white Guinea yam(Dioscorea rotundata). In vitro regeneration of Bambara groundnut (Vigna subterranea L.Verdc) via mersitem culture. In vitro micropropagation of medicinal plants.
- 58. Conclusion The ability toregenerate entire plants from cells or tissues has been invaluable to plant biotechnology due to the totipotent nature of plant cells. From the sole objective of demonstrating the totipotency of differentiated plant cells, the technique now finds application in both basic and applied researches in number of fields of enquiry. Plant tissue culture has generated intense interest among molecular biologists, plant breeders and commercial horticulturists. This technique should therefore be encouraged and supported by African countries as it could aid in improving food security in Africa.
- 60. References D.Dumet,A.Adeyemi,O.B.Ojuederie (2008). Yam invitrogenebanking. Genebank manual. http://www.iita.org/genebank/manual D.Dumet,A.Adeyemi,O.B.Ojuederie (2008).Cassava in vitro processing and the genebanking.Genebank manual. http://www.iita.org/genebank/manual HORT689/AGRO689 Biotechniques in Plant Breeding H.S Chawla .2002 Introduction to Plant Biotechnology 2nd edition. Oxford & IBH Publishing C./ Pvt. Ltd New Delhi India Murashige T. and Skoog F. (1962). A revised medium for rapid growth and bioassay withTobacco tissue culture. Physiologia plantarum 15: 473-497.