10.2 Plant tissue culture and micropropagation
7 min read•august 20, 2024
Plant tissue culture is a powerful technique for growing plant cells, tissues, or organs in sterile conditions. It enables rapid production of genetically identical plants, disease elimination, and genetic improvement. This method is widely used in horticulture, agriculture, and plant science research.
Micropropagation, a specific type of plant tissue culture, produces whole plants from small explants. It relies on plant cell totipotency and involves stages like preparation, establishment, , rooting, and acclimatization. This technique offers advantages like rapid multiplication but has challenges such as high costs and potential genetic instability.
Overview of plant tissue culture
- Plant tissue culture involves growing plant cells, tissues, or organs in a sterile environment on an artificial nutrient medium
- Enables the production of genetically identical plants (clones) from a single parent plant or explant
- Widely used in horticulture, agriculture, and plant science research for rapid propagation, disease elimination, and genetic improvement of plants
Principles of micropropagation
- Micropropagation is a specific type of plant tissue culture that involves the production of whole plants from small explants (pieces of plant tissue)
- Relies on the totipotency of plant cells, which is the ability of any living cell to regenerate into a whole plant under appropriate conditions
- Enables the production of large numbers of genetically uniform plants in a relatively short time and limited space
Stages of micropropagation
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Frontiers | A Micropropagation Protocol for the Endangered Medicinal Tree Prunus africana (Hook ... View original
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- Stage 0: Preparation of stock plants to provide healthy, disease-free explants
- Stage 1: Establishment of aseptic cultures by surface sterilization and plating of explants on nutrient media
- Stage 2: Multiplication of shoots or embryos through repeated subculturing on media with high cytokinin levels
- Stage 3: Rooting of shoots on media with high auxin levels to produce complete plantlets
- Stage 4: Acclimatization of plantlets to ex vitro conditions and transfer to soil or potting mix
Advantages vs disadvantages
- Advantages:
- Rapid multiplication of plants, especially those that are difficult to propagate by conventional methods (orchids, woody species)
- Production of through culture and virus indexing
- Preservation of genetic resources and rare or endangered species
- Facilitation of genetic improvement through somaclonal variation, somatic hybridization, and
- Disadvantages:
- High initial costs for laboratory equipment, facilities, and trained personnel
- Risk of genetic instability (somaclonal variation) due to long-term culture and exposure to plant growth regulators
- Difficulty in acclimatizing some species to ex vitro conditions, leading to high mortality rates
- Potential for spreading systemic diseases or mutations if not properly screened and tested
Techniques for establishing cultures
- Successful micropropagation depends on the establishment of aseptic cultures free from contaminating microorganisms
- Involves the selection of suitable explants, surface sterilization, and plating on appropriate culture media
Surface sterilization methods
- Chemical sterilants: Sodium hypochlorite (NaOCl), calcium hypochlorite (Ca(ClO)2), mercuric chloride (HgCl2), hydrogen peroxide (H2O2)
- Antibiotics: Gentamicin, streptomycin, rifampicin, used to control bacterial contamination
- Fungicides: Benomyl, nystatin, used to control fungal contamination
- Exposure time and concentration vary depending on the explant type and sensitivity to sterilants
Explant selection and preparation
- Suitable explants: Apical or axillary buds, shoot tips, nodal segments, embryos, leaves, roots
- Factors affecting explant choice: Genotype, age, physiological state, position on the plant
- Pretreatment of stock plants: Growth under controlled conditions, pruning, spraying with fungicides or insecticides
- Excision and trimming of explants to remove damaged or contaminated tissues and reduce explant size
Culture media components
- Culture media provide the necessary nutrients, energy sources, and growth regulators for the growth and development of plant tissues in vitro
- Composition varies depending on the species, explant type, and stage of micropropagation
Macronutrients and micronutrients
- Macronutrients: Nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), supplied as inorganic salts
- Micronutrients: Iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu), molybdenum (Mo), chlorine (Cl), cobalt (Co), nickel (Ni), supplied as inorganic salts or chelates
- Murashige and Skoog (MS) medium is the most widely used basal medium, containing optimal concentrations of macro- and micronutrients for many species
Plant growth regulators
- : Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), promote cell division, elongation, and rooting
- : Benzylaminopurine (BAP), kinetin, zeatin, thidiazuron (TDZ), promote cell division, shoot proliferation, and delay senescence
- Gibberellins: Gibberellic acid (GA3), promote stem elongation and break dormancy
- Abscisic acid (ABA): Regulates stomatal closure, induces dormancy, and inhibits growth
- Ethylene: Promotes fruit ripening, abscission, and senescence, inhibits shoot elongation
Organic supplements
- Vitamins: Thiamine (B1), pyridoxine (B6), nicotinic acid (niacin), myo-inositol, promote cell division and growth
- Amino acids: Glycine, glutamine, asparagine, provide reduced nitrogen and stimulate growth
- Complex organic extracts: Coconut water, casein hydrolysate, yeast extract, malt extract, provide vitamins, amino acids, and growth factors
- Gelling agents: Agar, gellan gum, phytagel, provide support for explants and prevent hyperhydricity
Types of plant tissue cultures
- Different types of cultures can be established depending on the explant type, media composition, and desired outcome
- Each culture type has specific applications in plant propagation, research, and biotechnology
Callus cultures
- Undifferentiated mass of proliferating cells, induced from various explants (leaves, stems, roots) on media with high auxin and low cytokinin levels
- Useful for producing secondary metabolites, somatic embryos, or genetically transformed cells
- Can be maintained indefinitely by regular subculturing on fresh media
- Examples: Production of paclitaxel (Taxol) from Taxus , vanilla flavoring from Vanilla planifolia callus
Suspension cultures
- Single cells or small cell clusters suspended in liquid media, obtained by agitating friable callus in media with high auxin levels
- Provide a homogeneous source of cells for studies on cell physiology, biochemistry, and genetic engineering
- Enable the production of secondary metabolites or recombinant proteins in bioreactors
- Examples: Production of shikonin from Lithospermum erythrorhizon cells, anthocyanins from Vitis vinifera cells
Organ cultures
- Cultures of isolated plant organs (roots, shoots, flowers, fruits) on media with specific growth regulator combinations
- Allow the study of organ development, physiology, and metabolism in a controlled environment
- Enable the production of virus-free plants through meristem culture and cryopreservation
- Examples: Propagation of potato through nodal cultures, production of pathogen-free citrus through shoot tip grafting
Factors affecting culture growth
- The growth and development of plant tissue cultures are influenced by various physical and chemical factors in the culture environment
- Optimization of these factors is crucial for successful micropropagation and production of high-quality plants
Physical factors
- Temperature: Affects cell division, elongation, and differentiation, optimal range is 20-28°C for most species
- Light: Regulates photosynthesis, morphogenesis, and secondary metabolite production, optimal intensity and photoperiod vary by species and culture stage
- Humidity: Influences transpiration, nutrient uptake, and hyperhydricity, maintained at 40-70% relative humidity in culture vessels
- Aeration: Provides oxygen for respiration and removes ethylene, achieved through culture vessel design, media solidification, and forced ventilation
Chemical factors
- pH: Affects nutrient availability, enzyme activity, and cell growth, optimal range is 5.5-6.0 for most species
- Carbohydrate source: Provides energy and carbon skeletons for growth, sucrose is the most common, but glucose, fructose, or maltose may be used
- Mineral nutrition: Balanced supply of macro- and micronutrients is essential for cell growth and differentiation, deficiencies or toxicities can impair culture performance
- Plant growth regulators: Specific combinations and concentrations of auxins, cytokinins, gibberellins, and other hormones regulate cell division, differentiation, and
Applications of micropropagation
- Micropropagation has numerous applications in agriculture, horticulture, forestry, and plant science research
- Enables the rapid multiplication of superior genotypes, conservation of genetic resources, and genetic improvement of crops
Commercial plant production
- of ornamental plants (orchids, roses, chrysanthemums), fruit crops (banana, strawberry, pineapple), and forest trees (eucalyptus, pine, teak)
- Production of disease-free planting materials for crops prone to viral infections (potato, sugarcane, cassava)
- Multiplication of genetically engineered plants with improved traits (herbicide resistance, insect resistance, enhanced nutritional value)
Conservation of rare species
- In vitro conservation of endangered or threatened plant species through slow-growth storage or cryopreservation of shoot tips, embryos, or callus
- Reintroduction of micropropagated plants into natural habitats to increase population size and genetic diversity
- Establishment of in vitro gene banks for long-term preservation of plant genetic resources
Genetic improvement of crops
- Somaclonal variation: Selection of novel traits (disease resistance, stress tolerance, altered morphology) from plants regenerated from callus or cell cultures
- Somatic hybridization: Fusion of protoplasts from different species or genera to create interspecific or intergeneric hybrids with desired traits
- Genetic transformation: Introduction of foreign genes into plant cells using Agrobacterium tumefaciens, particle bombardment, or electroporation, followed by regeneration of transgenic plants
Challenges in micropropagation
- Despite its numerous advantages, micropropagation also faces several challenges that can limit its efficiency and commercial application
- Addressing these challenges requires a combination of technical, scientific, and managerial solutions
Contamination and culture losses
- Microbial contamination (bacteria, fungi, yeast) is a major cause of culture losses and reduced efficiency in micropropagation
- Sources of contamination: Explants, culture media, laboratory environment, personnel
- Prevention strategies: Strict aseptic techniques, surface sterilization of explants, use of antibiotics or fungicides, regular monitoring and disposal of contaminated cultures
Somaclonal variation
- Genetic or epigenetic changes in plants regenerated from tissue cultures, resulting in altered morphology, physiology, or performance
- Causes: Chromosomal rearrangements, point mutations, DNA methylation, activation of transposable elements
- Consequences: Loss of genetic fidelity, reduced yield or quality, increased susceptibility to pests and diseases
- Mitigation strategies: Minimizing the duration of culture, using low concentrations of growth regulators, selecting stable genotypes, and thorough field testing of micropropagated plants
Cost and scalability issues
- High initial investment in laboratory infrastructure, equipment, and skilled personnel
- Labor-intensive and time-consuming process, especially for species with low multiplication rates or difficult rooting
- Need for specialized facilities for acclimatization and hardening of micropropagated plants
- Challenges in scaling up production to meet commercial demands while maintaining quality and uniformity
- Strategies to reduce costs: Automation of culture processes, use of low-cost media components, optimization of culture protocols, and integration with conventional propagation methods
Key Terms to Review (18)
Aseptic technique: Aseptic technique is a set of practices aimed at preventing contamination by unwanted microorganisms during laboratory procedures. This method is crucial in plant tissue culture and micropropagation, where maintaining a sterile environment ensures that the desired plant cells can grow without interference from pathogens or other contaminants. It involves specific actions like sterilizing equipment, using sterile media, and working in a clean environment to promote successful cultivation and propagation.
Auxins: Auxins are a class of plant hormones that play a crucial role in regulating plant growth and development, particularly by influencing cell elongation, apical dominance, and responses to light and gravity. These hormones are essential for coordinating various physiological processes in plants, including growth patterns and developmental stages.
Callus: A callus is a mass of undifferentiated plant cells that forms at the site of injury or during tissue culture processes. This cellular mass has the unique ability to develop into various types of plant tissues, making it essential for regeneration and propagation in plant tissue culture and micropropagation techniques. Callus formation is a crucial step in producing new plants from small tissue samples, enabling efficient plant breeding and conservation efforts.
Clonal propagation: Clonal propagation is a method of asexual reproduction in plants that produces genetically identical offspring from a single parent plant. This technique is widely used in horticulture and agriculture because it allows for the rapid multiplication of plants with desirable traits, ensuring uniformity and consistency across the propagated individuals. The process often involves techniques such as cuttings, layering, and tissue culture to create new plants that maintain the genetic characteristics of the original plant.
Contamination risk: Contamination risk refers to the potential for unwanted microorganisms, such as bacteria and fungi, to enter and proliferate in plant tissue culture environments, which can lead to compromised cultures and failed experiments. This risk is particularly significant in plant tissue culture and micropropagation, where sterile conditions are crucial for the success of growing plants from small tissue samples. Managing this risk is vital for maintaining the health of cultures and achieving reliable results.
Cytokinins: Cytokinins are a class of plant hormones that promote cell division and growth, playing a critical role in various plant developmental processes. They help regulate the growth of shoots and roots, influence the aging process of plants, and can delay senescence, which is the aging and death of plant cells. Their effects can be seen in tissue culture and micropropagation, where they are used to stimulate shoot development and enhance propagation success.
Disease-free plants: Disease-free plants are specimens that have been cultivated without the presence of pathogens, such as bacteria, viruses, or fungi, which can negatively affect their growth and health. These plants are crucial for agricultural practices as they ensure higher yields and better quality produce, and their cultivation often relies on techniques such as plant tissue culture and micropropagation to maintain genetic integrity and eliminate diseases.
Genetic engineering: Genetic engineering is the direct manipulation of an organism's DNA to alter its characteristics, often involving the addition, deletion, or modification of specific genes. This technique allows scientists to create organisms with desirable traits, enhancing agricultural productivity, disease resistance, and overall resilience. Its applications span various fields, including agriculture and biotechnology, making it a crucial tool for addressing global challenges like food security and environmental sustainability.
Growth medium: A growth medium is a substance or mixture that provides the essential nutrients and environment for plant cells to grow and multiply in vitro. It plays a crucial role in plant tissue culture and micropropagation by supplying the necessary elements such as water, minerals, vitamins, and hormones to support cellular development and differentiation.
Initiation: Initiation refers to the process that marks the beginning of a specific developmental stage in plant tissue culture, where cells or tissues are induced to proliferate and differentiate into new plant structures. This process is critical in plant tissue culture and micropropagation as it sets the stage for regeneration and growth of whole plants from small tissue samples, enabling large-scale production and preservation of plant species.
Leaf explant: A leaf explant is a small piece of leaf tissue used in plant tissue culture for the purpose of regenerating new plant cells or tissues. This technique allows for the propagation of plants in a controlled environment, enabling researchers and horticulturists to produce clones of plants efficiently. Leaf explants can be used to develop entire plants from a single leaf segment, making them an essential tool in micropropagation.
Light conditions: Light conditions refer to the availability and intensity of light in a given environment, which plays a crucial role in the growth and development of plants. These conditions can vary widely depending on factors such as the time of day, season, and geographical location. Understanding light conditions is essential in plant tissue culture and micropropagation, as they influence photosynthesis, cell growth, and overall plant health.
Meristem: A meristem is a region in plants consisting of undifferentiated cells that have the ability to divide and differentiate into various types of specialized tissues. These cells are crucial for growth and development, as they enable the plant to produce new tissues, organs, and even entire plants through processes like tissue culture and micropropagation.
Multiplication: Multiplication is a biological process that refers to the rapid production of multiple identical copies of a plant or plant part through methods like tissue culture. This technique enables the growth of new plants from small tissue samples, which are nurtured in a controlled environment, leading to the development of numerous genetically identical individuals. It's a vital part of modern plant propagation, allowing for the preservation of desirable traits and the mass production of plants for agriculture and horticulture.
Organogenesis: Organogenesis is the process by which specific tissues and organs develop from the undifferentiated cells of an embryo or a tissue culture. This phenomenon is crucial in plant development, where it allows for the formation of structures like roots, shoots, and leaves. Understanding organogenesis is essential in plant tissue culture and micropropagation, as it enables scientists to manipulate growth patterns and generate new plants from small cell samples.
Somatic embryogenesis: Somatic embryogenesis is a process where somatic cells, which are non-reproductive cells, develop into embryos and ultimately form whole plants. This method allows for the production of genetically identical plants from a single somatic cell, making it a vital technique in plant tissue culture and micropropagation. It is particularly significant for producing disease-free plants and can be used for the rapid propagation of desirable plant varieties.
Stem explant: A stem explant is a piece of plant tissue, specifically from the stem, that is removed and used in plant tissue culture to regenerate new plants. This technique allows for the propagation of plants under controlled conditions, providing a means to produce clones that maintain the genetic identity of the parent plant. Stem explants can be manipulated to encourage growth and development into full plants, making them a vital component in micropropagation processes.
Subculture: In the context of plant tissue culture and micropropagation, a subculture refers to the process of transferring a small portion of a cultured plant tissue or cells to a new growth medium for further growth and multiplication. This technique allows for the maintenance and propagation of plant tissues, ensuring that the desirable traits are preserved while promoting healthy growth in a controlled environment.