Introduction to: Plant Tissue Culture
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This document provides an overview of plant tissue culture techniques. It defines plant tissue culture as growing plant cells, organs or tissues in a sterile environment with nutrient media. The key requirements for plant tissue culture are appropriate explant tissue, a suitable growth medium, aseptic conditions, growth regulators, and frequent sub-culturing. Plant tissue culture has advantages over working with intact plants like enabling large-scale growth and genetic modification. The document discusses regeneration methods like shoot regeneration and somatic embryogenesis that allow developing whole plants from cultured cells or tissues. It also covers the stages of micropropagation including multiplication, rooting, and acclimatization of plantlets.
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Introduction to: Plant Tissue Culture
- 1. Introduction to: Plant Tissue Culture Jaskaran Kaur Arora L-2016-A-19-D Biotech 606
- 2. CONTENTS Plant Tissue Culture • Definition • Flow chart of general technique • Advantages over intact plant Regeneration • Introduction • Shoot regeneration • Somatic embryogenesis Plant Multiplication • Micropropagation stages • Limitation
- 4. Definitions: • “Tissue culture” widely for in vitro culture of cells, tissues as well as organs • Plant Tissue Culture is a technique of growing plant cells, organs, seeds or other plant parts in a sterile environment on nutrient medium.
- 5. Basics of Plant tissue culture • In vitro cultivation primarily to solve two basic problems: 1. To keep plants and organs free from microbes 2. To ensure desirable development in cells and organs by providing suitable nutrient media & other env. conditions. Area of active research
- 6. What conditions do plant cells need to multiply in vitro? Tissue culture has several critical requirements: Appropriate tissue A suitable growth medium Aseptic (sterile) conditions Growth regulators Frequent sub-culturing
- 7. Appropriate tissue (Explant) • Explants: Cell, tissue or organ of a plant that is used to start in vitro cultures. • Most commonly: axillary buds and meristems • The explants must be sterilized to remove microbial contaminants Chemical Conc. Duration (min) Bromine water 1-2 % 5-10 Calcium hypochlorite About 10% 5-30 Hydrogen peroxide 10-12 % 10-15 Mercuric chloride 0.1-1.0 % 5-15 Sodium hypochlorite 2 % 5-30 Antibiotics 4-50 mg/L 30-60
- 8. Growth medium • A nutrient medium is defined by its mineral salt composition, carbon source, vitamins, plant growth regulators and other organic supplements. Composition: Inorganic nutrients Vitamins Carbon source Growth Regulators Complex organic additives
- 9. Yeast Extract Coconut Milk Casein hydrolysate Malt Extract • Inositol •Thiamine •Pyridoxine •Nicotinic Acid Vitamins Carbon Source •Sucrose •Glucose •Maltose •Galactose • Mannose •Starch Organic Additives
- 10. Sterile conditions • All materials: vessels, instruments, medium, plant material. etc must be free from microbes • Following approaches: Flame sterilization Autoclaving Filter Sterilization Wiping with 70 % ethanol Surface sterilization
- 12. Flow chart of plant propagation by: tissue culture method
- 13. Advantages of tissue culture OVER intact plants 1. Can grow plant cells in liquid culture on a large scale—Bioreactor 2. Dihaploid plants production from haploid cultures shortens the time taken to achieve uniform homozygous lines 3. The crossing of distantly related species by protoplast isolation and somatic fusion help in transfer and expression of novel genes 4. Cell selection increases the potential number of individuals in a screening program 5. Micropropagation allows the production of large numbers of uniform individuals of species from limited starting material 6. Genetic transformation of cells enables very specific information to be introduced into single cells which can then be regenerated
- 14. REGENERATION OF TISSUE CULTURE Part II:
- 15. Plant regeneration • The process of growing an entire plant from a single cell or group of cells • Possible because plant cells can be made totipotent using hormones • Differentiated tissue: stem, leaves, roots etc. • Undifferentiated cells are totipotent: can become whole plant by differentiating into whole plant
- 17. Plant Regeneration System Plant regeneration Organogenesis--Shoots or roots are induced to differentiate from a cell or cell clusters (Shoot regeneration will be discussed) Somatic embryogenesis--New plants are formed from somatic embryos. Somatic embryos are formed in plant tissue culture from plant cells that are not normally involved in the development of embryos, i.e. ordinary plant tissue
- 18. Shoot Regeneration • First report by :White in 1939 in tobacco tissue culture • Shoot buds usually arise from group of meristematic cells called meristemoids/nodules • Meristemoids leaf primordia and apical meristem • Arise in areas that accumulate starch • GA3 inhibit shoot regeneration by interfering with starch accumulation
- 19. Shoot induction Shoot differentiation and development Events during shoot regeneration Morphogenic competence acquisition Phase Developmental determination phase Commitment is irreversible
- 20. Factors affecting shoot bud regeneration Growth regulators Explant genotype Physiological state of explant Physical factors Electrical Stimulation
- 21. Somatic Embryogenesis • Somatic embryo is an embryo derived from a somatic cell, other than zygote. • Somatic embryogenesis is defined as the process of development of a bipolar structure like zygotic embryo from a non-zygotic somatic cell • Doesn’t have vascular connections • Reported in 1968 independently by Steward and Reinert in carrot
- 22. Developmental pattern of SEs Direct SE regeneration is most likely to occur from ovules, zygotic embryos and young seedlings
- 24. Gene Gene product Function CHB-2 Homoeoprotein Vascular element diff. EP2 Lipid transfer protein (LTP) Acquisition of embryogenic potential AlLTP1 LTP Protoderm formation EP3 Extracellular endochitinase Protoderm formation TS11 Arabinogalactan proteins (AGPs) Globularheart shaped Some of the Genes involved in SE..
- 25. S.N Characteristic Shoot Bud Somatic Embryo 1 Origin Many cells Single cell 2 Polarity Unipolar Bipolar 3 Vascular connection with callus/explant Present Absent 4 Separation from callus/explant Not easily separated unless cut off Easily separated as radicular end is cutinized Comparison between shoot bud & SE
- 27. Introduction • Micropropagation is the process of rapidly multiplying stock plant material to produce large number of progeny plants using plant tissue culture methods • Achieved by following processes: Proliferation of axillary buds Induction of adventitious buds Organogenesis Somatic Embryogenesis • For axillary bud proliferation: Pre-existing meristem cultured
- 28. • SAM is the portion lying distal to youngest leaf primordium (100 μm dia and 250 μm in length) • Shoot tip apical meristem + 1-3 young leaf primordia (=500 μm) • If objective is rapid propagation size is not important • If objective is virus free minimum surrounding tissue Meristem culture
- 29. Meristem culture • Shoot tips of 0.5 mm or longer grow into shoots on GR-free media (low levels of auxin/or cytokinin is preferred) • Without leaf primordia culture auxin is essential in most cases and cytokinin in some cases (as meristematic dome doesn’t produce its own auxin and cytokinins) 2,4-D avoided GA3 is often used Leaf primordia provides app. amount of auxin and cytokinin
- 30. 0 • Selection and preparation of mother plants 1 • Culture initiation 2 • Multiplication 3 • Rooting of shoots 4 • Transfer to soil Stages of Micropropagation
- 31. • Identification and preparation of mother plants more responsive explants • Stock plants under controlled conditions (glass house)good • Overhead irrigation should be avoided • Reduce level of contamination • To increase responsiveness light, temp, GR • “Rejuvenation” in tree species 0-Preparation of mother plants
- 32. • Surface sterilization of explants and establishing them in vitro • Main feature detection and elimination/control of contamination • GR-free medium used • In case of contamination antibiotic or fungicide may be added • Most commonly used explants are organs, shoot tips, nodal segments. 1-Culture initiation
- 33. • Most crucial stage as it determines the rate at which plantlets are formed • Multiplication by: Enhanced proliferation of axillary shoot buds Induction of adventitious buds, bulbs, etc. Somatic embryogenesis (SE) • Defined culture medium 2-Multiplication
- 34. • GR-free/ auxin stimulation • On agar medium in vitro rooting • Directly on potting mix after treating ends with auxin solution ex vitro/ in vivo rooting 3-Rooting of shoots Rooting and soil transfer stages are combined Structurally and functionally better roots Better rooting to difficult root species No risk of root damage during transfer to soil Saving of labour and media reagents
- 35. 4-Transfer to soil Rooted shoots are removed from medium Agar sticking to roots washed with tap water Transplanted into plastic cups ↓ High Humidity: Fog Mist Clear cups
- 36. Ex vitro transfer Photoheterotrophic state TO Photoautotrophic Low humidity Higher Irradiance To make them capable of surviving...
- 37. 1. Modified nutrient media and culture condition 2. Chemical additives in culture media 3. Co-culture with microorganisms . . . Hardening/Acclimatization ...1 ↑ aeration ↑ light intensity ↑ CO2 level ↓ Sucrose level Favour photoautotrophy ...2 Polyethylene glycol Paclobutrazol Cuticular Biosynthesis ...3 Bacterization with Pseudomonas Root Colonization with endomycorrhia ↑ Lignin content, ↑ stomatal function, ↑ tolerance to dehydration
- 38. Choice of Route for Micropropagation When all three (axillary, adv, SE) are available Axillary is preferable Chimeras (two genetically different tissues) Axillary Desirable feature due to virus as in geranium “crocodile” variety adv shoot bud regeneration Easier, faster and more practicable route of micropropagation Golden netting in the leaf veins of Geranium Bulblet regeneration from bulb scale in lilies
Editor's Notes
- Area of active research: relies mainly on the manipulation of culture medium especially GR and to much lesser extent on other factors
- Tissue culture has several critical requirements: Appropriate tissue A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid. Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can overrun a culture. Growth regulators - in plants, both auxins & cytokinins. Frequent subculturing to ensure adequate nutrition and to avoid the build-up of waste metabolites
- This is usually done by chemical surface sterilization with an agent for a duration that will kill pathogens w/o injuring the plant cells
- When an explant is isolated, it is no longer able to receive nutrients or hormones from the plant, and these must be provided to allow growth in vitro. The composition of the nutrient medium is for the most part similar, although the exact components and quantities will vary for different species and purpose of culture. Types and amounts of hormones vary greatly. In addition, the culture must be provided with the ability to excrete the waste products of cell metabolism. This is accomplished by culturing on or in a defined culture medium which is periodically replenished.
- N in the form of amino acids (glutamine, asparagine) and nucleotides (adenine) - Organic acids: TCA cycle acids (citrate, malate, succinate, fumarate), pyruvate - Complex substances: yeast extract, malt extract, coconut milk, protein hydrolysate - Activated charcoal is used where phenol-like compounds are a problem, absorbing toxic pigments and stabilizing pH. Also, to prevent oxidation of phenols PVP (polyvinylpyrrolidone), citric acid, ascorbic acid, thiourea and L-cysteine are used.
- protoderm. a thin outer layer of the meristem in embryos and growing points of roots and stems, which gives rise to the epidermis.
- Shoot apical meristems produce one or more axillary or lateral buds at each node. When stems produce considerable secondary growth, the axillary buds may be destroyed. Adventitious buds may then develop on stems with secondary growth.