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, multiplication, 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

• 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 disease-free plants through meristem culture and virus indexing
  • Preservation of genetic resources and rare or endangered species
  • Facilitation of genetic improvement through somaclonal variation, somatic hybridization, and genetic engineering
• 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

• Auxins: 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
• Cytokinins: 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 callus, 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 organogenesis

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

• Clonal propagation 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