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PLANT TISSUE CULTURE
- 1. PLANT TISSUE CULTURE AND ITS APPLICATIONS Hafiz Muzammil Nisar MPhil Biotechnology Bahaudin Zakariya University, Multan.
- 2. Contents: • History • Introduction • Tissue culture • Nutrient medium • Fundamental Principles of plant tissue culture • Plant tissue culture Techniques • Micropropagation • Somatic cell Genesis • Transgenic Plants • Major steps of Tissue Culture • Initiation phase • Multiplication phase • Root formation phase • Types of Tissue culture • Cell culture • Callus culture • Protoplast culture • Organ culture • Seed culture • Embryo culture • Meristem culture • Anther culture • Applications of Tissue Culture • Conclusion
- 3. History • Haberlandt, Father of plant tissue culture. cultured single cells on Knop’s salt solution. • Hanning(1904) Embryo culture of selected crucifers. • Robbins(1922), in vitro culture of root tips. • Muir(1953) isolation and culture of single plant cells. • Skoog and Miller(1957) hormonal control in tissue culture. • Reinert and Steward(1958) report somatic embryogenesis.
- 4. History • E C Cocking (1960) Isolation of protoplasts by enzymatic degradation method. • Murashige and Skoog (1962) Development of MS media. • Guha and Maheshwari (1964) Production of First haploid plant by anther culture
- 5. Plant Tissue Culture • The growth or regeneration of plant cells, tissues, organs or whole plants in artificial medium under aseptic conditions. • In this technique use plant parts or cells called Explant. INTRODUCTION: • The most widely used artificial nutrient medium is MS medium.
- 6. Fundamental Principles of Plant Tissue Culture Plant tissue culture depends upon; • Totipotency, ability of plant cells to regenerate into whole new plant • Plasticity, ability of plants to alter their metabolism, growth and development to best suit their environment Plant cell Tissue Callus Plant organ Embryo New plant
- 7. Plant Tissue Culture Techniques: 1. Micropropagation: • Developing high- quality clonal plants. • Provide rapid and large scale propagation of new genotypes. • Steps: • Sterilization and introduction • Shoot production • Root production • Transfer to soil
- 8. 2. Somatic cell genetics: • It is used mostly in terms of haploid production and somatic hybridization. • Isolation of protoplast • Fusion by using chemicals • Plating of fused protoplast • Selection • Steps:
- 9. 3. Transgenic plants: • They are identified as a class of genetically modified organism (GMO). • Transgenic plants are plants that have been genetically engineered, uses recombinant DNA techniques to create plants with new characteristics. • The complete process is described as:
- 10. Major Steps of Tissue Culture: • Initiation Phase (Stage 1) • Multiplication Phase (Stage 2) • The in vitro plant material is introduced in to the medium. • Proliferation of the tissue and the production of multiple shoots. • The tissue of interest is obtained, sterilized and introduced.
- 11. Major Steps of Tissue Culture: • Roots are formed. • Here, hormones are required in order to induce rooting such as “Auxin”. • Consequently complete plantlets. • Root formation (Stage 3)
- 13. Cell culture: • The culture of isolated individual cells. • Carried out in dispersion medium. Callus culture: • The growth of callus from explant by providing appropriate conditions. • Darkness and solid medium gelled by agar stimulate callus formation.
- 14. Protoplast culture: • The protoplast culture is aimed to develop genetically transformed plant. • The cell in which the cell wall has been removed. • In this, isolated protoplasts are cultured on a suitable medium under the aseptic condition.
- 15. Organ culture: • Isolated plant organs (Shoot, root, leaf, and flower) are used as explant. • The organ culture may be organized or unorganized. • New growth (differentiated structures) continues given that the organ retains its: • Physiological features, • Provide information on patterns of growth, • As well as development.
- 16. Seed culture: • For this method, explants are obtained from an in-vitro derived plant. • Introduced into an artificial environment. • Where they get to proliferate. • Seed culture is primarily used for plants such as orchids
- 17. Embryo Culture: • For embryo culture, the ovule, seed or fruit from which the embryo is to be obtained is sterilized. • Culturing them in nutrient media, help in developing viable seedlings. • Embryo culture is the isolation and growth of mature or immature embryo in-vitro.
- 18. Meristem culture: • A meristem is a localized group of cells, which are actively dividing and give rise to permanent tissue. • The apical meristem of shoots is cultured. • To get the disease-free plants.
- 19. Anther Culture: • Anthers of some plants are cultured on a suitable medium to produce haploid plants, it is called anther culture. • Separated anthers from flowers. • Cultured on a suitable medium. • This technique was first used in India to produce haploids of Datura.
- 20. Applications of Plant Tissue Culture:
- 21. Clonal Propagation: • Useful tool to get a large population of plant species having desirable traits. • This technique is very much used in horticulture and silviculture—orchids and many fruit plants. • Large scale production of plants of same genetic stock.
- 22. Secondary Metabolites: • Secondary metabolites produced by plant cell cultures are rather small in amount. • By clonal selection the particular high yielding clone of cells can be isolated. • Production of many useful compounds can be done by plant cell culture.
- 23. Genetic Variability: • The chromosomal instability in the cultured cells play an important role in polyploidization of cells and genetically variable plants can be raised. • Such kind of variations may show some useful characters such as: • Different kinds of morphological variations in leaf and flowering.
- 24. Somatic Embryogenesis and Synthetic Seed: • Direct or indirect somatic embryogenesis may be achieved from: • Pro-embryonic cell of the direct explant • The embryoids developed within the callus tissue. • The application of this technique is: • the mass production of adventitious embryos which ultimately develop into complete plantlet.
- 25. Haploid Plants: • Haploid plants can be obtained through anther or pollen culture (androgenesis). • These haploids produced homozygous diploid or polyploid lines. • Colchicine treatment within a very short period.
- 26. Somatic Hybrids: • Somatic hybridization, using isolated somatic protoplasts is a new tool to make the wide hybridization successful. • Obtain somatic hybrid plants between sexually compatible and incompatible plants. • Production of cybrid is also of immense importance in the plant breeding program.
- 27. Transgenic Plants: • The genetically modified (GM) plants, in which a functional foreign gene has been incorporated by biotechnological method. • A number of transgenic plants have been produced carrying genes for different traits. • The direct DNA uptake through protoplast is the most ideal method.
- 28. Germplasm Conservation: • Many of the important crop species produce recalcitrant seeds. • Plant tissue culture may be applied for this purpose. • Mainly the plant species which are endangered, needed to be conserved by ex-situ method of germplasm conservation.
- 29. Conclusion: • Useful to human kind and ecosystem. • Hope to end world hunger. • Performed with cautions to minimize the risks. • Researcher must be ethical. • Use for cloning purposes and increase yield.
- 30. References: • https://www.nature.com/subjects/tissue-culture • https://www.studyread.com/plant-tissue-culture-techniques/https://lastdna.com/plant-tissue-culture/#medium • https://www.researchgate.net/figure/Conversion-of-microspore-derived-embryos-to-plantsa-b-c-Consecutive-stages- during_fig11_230628977 • https://www.sciencedirect.com/science/article/pii/S026121941100233X • https://agrihunt.com/articles/breeding-and-genetics/plant-regeneration-techniques/ • http://www.biologydiscussion.com/plant-breeding/micro-propagation/micro-propagation-methods-and-stages-biotechnology/61317 • http://www.biologydiscussion.com/cell/plant-cell/top-10-applications-of-plant-cell-and-tissue-culture-biotechnology/61263 • https://www.slideshare.net/maryamriaz16/somatic-hybridization-in-higher-plants
Editor's Notes
- This is a method of biological research in which fragments of tissue from an animal or plant are transferred to an artificial environment in which they can continue to survive and function. inorganic salts (both micro and macro elements), a carbon source (usually sucrose), vitamins, amino acids, growth regulators (e.g. auxins and cytokinin and gibberellins).
- Fig 1: banana plantlets transfer to soil produced by micropropagation.
- PEG, sodium nitrate, calcium ions.
- in order to prevent any microorganism from negatively affecting the process. It is during this stage that the tissue is initiated in to culture. Here, the medium is composed of appropriate components for growth including regulators and nutrients.
- Callus is the unorganized and undifferentiated dividing mass of cells. Darkness is required as light can encourage differentiation of the callus.
- Somatic hybridization of two plant species through protoplast fusion in done by protoplast culture.
- In the event that a plant material is used directly for this process, then it has to be sterilized to prevent tissue damage and ensure optimum regeneration.
- In some plants, seed dormancy may be due to chemical inhibitors or mechanical resistance etc. Embryo culture may involve the use of a mature of immature embryo. Whereas mature embryos for culture are essentially obtained from ripe seeds, immature embryo (embryo rescue) involves the use of immature embryos from unripe/hybrid seeds that failed to germinate. In doing so, the embryo is ultimately able to produce a viable plant. For embryo culture, the ovule, seed or fruit from which the embryo is to be obtained is sterilized, and therefore the embryo does not have to be sterilized again.
- Silviculture is the art and science of controlling the establishment, growth, composition, and quality of forest vegetation for the full range of forest resource objectives. in the plants which have long seed dormancy, tree species,
- Production of many useful compounds like alkaloids, Steroids (Diosgenin), Glycosidic compounds and many other essential oils (Jasmine), flavoring and coloring agents (saffron) can be done by plant cell culture. This aim can be achieved by selection of specific cells producing high amount of desired compounds and development of a suitable medium.
- Flowering time, plant height, lodging resistance, yield, nutrient content and
- Synthetic seed production, germination and field transfer of synthetic seeds of S. chirayita. (a)-encapsulated torpedo shaped somatic embryos in 100 mM CaCl 2 solution (bar = 0.5 cm); (b)-synthetic seeds produced using torpedo shaped somatic embryos (bar = 0.5 cm); (c)-germination of a synthetic seed on MS medium after 30 days of culture (bar = 1.0 cm); (d)-cluster of plantlets derived from a synthetic seed (bar = 1.2 cm); (e)-complete rooted plant from synthetic seed (bar = 1.0 cm); (f)-acclimatized plant after transfer to soil in ex vitro conditions. Ginseng transformation by Agrobacterium tumefaciens for the transgenic adventitious root induction.
- Colchicine is a toxic chemical that is often used to induce polyploidy in plants. Basically, the colchicine prevents the microtubule formation during cell division, thus the chromosomes do not pull apart like they normally do. The end result is a cell that now has double the number of chromosomes that it would normally have.
- This technique has been mainly used for introgression many useful criteria from the wild genotype to cultivated crop variety. Cybrid-the fusion between two protoplasts—one partner with nucleus and another partner with cytoplasm,
- like insect resistance, herbicide tolerance, delayed ripening, increased amino acid and vitamin content, improved oil quality, etc. The direct DNA uptake through protoplast is the most ideal method for production of transgenic plants. Any gene of interest that may be of eukaryotic or prokaryotic origin can be used for this purpose but should be expressed.
- Recalcitrant seeds (subsequently known as unorthodox seeds) are seeds that do not survive drying and freezing during ex-situ conservation and vice versa