Introduction
Plant tissue culture is a technique used to propagate plants under sterile conditions by culturing plant cells, tissues, or organs on nutrient media. This method is widely employed in plant science research, agriculture, and horticulture for various purposes such as micropropagation (rapid clonal propagation), germplasm conservation, and genetic modification.
History of Plant Tissue Culture
Early Beginnings (1902-1930s):
The early history of plant tissue culture is marked
by pioneering experiments and discoveries that laid the groundwork for modern
techniques. Here are the key milestones and contributions during this period:
1. Gottlieb Haberlandt (1902):
o
Conceptual Foundation: Haberlandt, an Austrian botanist, is often
considered the father of plant tissue culture. He first proposed the idea that
plant cells are totipotent, meaning that a single cell has the potential to
develop into an entire plant. This concept is fundamental to plant tissue
culture.
Initial Experiments: Haberlandt attempted to culture isolated plant cells and tissues in
vitro (outside the living organism) using simple nutrient solutions. Although
he did not successfully get the cells to divide, his pioneering work
provided a theoretical foundation for future research.
2. Philip R. White (1930s):
o
Tomato Root Culture: In the 1930s, American botanist Philip R. White successfully cultured
tomato root tips, demonstrating that plant tissues could be maintained and
grown in a nutrient medium outside the plant body. This was a significant
breakthrough as it showed the practical feasibility of plant tissue culture.
o
Continued Research: White's work on root culture laid the groundwork for more advanced
studies in tissue culture. He continued to refine the techniques and media
required for successful tissue culture.
Development Phase (1940s-1960s):
The development
phase of plant tissue culture from the 1940s to the 1960s was characterized by
several significant breakthroughs and advancements. This period saw the
refinement of techniques, the discovery of essential plant growth regulators,
and the development of nutrient media that laid the foundation for modern plant
tissue culture.
Key Contributions
1. Discovery of Plant Growth Regulators:
o Folke Skoog and Carlos O. Miller (1950s):
Cytokinins: Folke Skoog and his graduate student Carlos O. Miller discovered cytokinins, a class of plant growth regulators that promote cell division. They identified kinetin as a naturally occurring cytokinin. The discovery of cytokinins was crucial as it helped scientists understand how to regulate and control cell division and differentiation in plant tissue cultures.
Role of Auxins: During this period, auxins
(another class of plant hormones) were also recognized for their role in
promoting cell elongation and root formation. The interplay between cytokinins
and auxins became a central focus in tissue culture research, allowing
scientists to manipulate plant tissues more effectively.
2. Development of Nutrient Media:
o Murashige and Skoog (1962):
MS Medium: Toshio Murashige and Folke Skoog developed the Murashige and Skoog (MS) medium in 1962, which became one of the most widely used nutrient media in plant tissue culture. The MS medium provided a balanced mixture of macro- and micronutrients, vitamins, and growth regulators, enabling the successful culture of a wide variety of plant tissues.
Other Media Formulations: Alongside the
MS medium, other media formulations were developed to suit specific plant
species and tissue types. These included Gamborg’s B5 medium and Woody Plant
Medium (WPM), each tailored to support the growth of different plant tissues.
Modern Era (1970s-present):
The modern era of
plant tissue culture, from the 1970s to the present, has been characterized by
significant technological advancements, the development of new techniques, and
a broadening scope of applications in research, agriculture, and industry
Key Developments and Techniques
1. Advancements in Genetic Engineering:
o Agrobacterium-Mediated Transformation: In the 1980s,
the use of Agrobacterium tumefaciens, a soil bacterium, became a standard
method for introducing foreign genes into plant cells. This technique involves
the insertion of the desired gene into the T-DNA region of the Agrobacterium
plasmid, which is then transferred into the plant genome.
o Biolistics (Gene Gun): The gene gun or
biolistic particle delivery system was developed as an alternative method for
gene transfer, particularly useful for plants that are less susceptible to
Agrobacterium. This method involves bombarding plant tissues with tiny
particles coated with DNA.
2. Development of Advanced Culture Techniques:
o Somatic Embryogenesis: Techniques for
somatic embryogenesis, where somatic cells develop into embryos and
subsequently into whole plants, have been refined. This method is widely used
for clonal propagation and genetic transformation.
o Protoplast Culture and Fusion: Protoplast
culture involves the isolation and culture of plant cells without cell walls.
Protoplast fusion techniques allow the combination of genetic material from
different species, leading to the creation of somatic hybrids.
3. Cryopreservation:
o Long-term Storage: Cryopreservation
techniques have been developed for the long-term storage of plant tissues,
cells, and embryos at ultra-low temperatures. This is crucial for the
conservation of genetic resources and germplasm.
4. Bioreactor Systems:
o Large-Scale Propagation: The development
of bioreactor systems has enabled the large-scale production of plant tissues
and cells. These systems provide controlled environments for the efficient and
scalable propagation of plants.
Scope of Plant Tissue Culture
Research
Plant tissue culture plays a crucial
role in plant research, offering various techniques to study plant physiology,
genetics, biochemistry, and more.
1. Studying Plant Physiology:
Cellular Processes:
o Cell Division and Differentiation: Tissue culture
allows researchers to observe how plant cells divide and differentiate into
various tissues and organs. By manipulating the culture conditions (e.g.,
nutrient composition, growth regulators), scientists can study the factors
influencing these processes.
o Metabolism: In vitro cultures provide a
controlled environment to study metabolic pathways and the effects of different
nutrients, hormones, and environmental conditions on plant metabolism.
o Signal Transduction: Researchers can
investigate how plants perceive and respond to external signals, such as light,
temperature, and stress, by using tissue cultures to control and monitor these
variables precisely.
2. Developmental Biology:
Embryogenesis: Somatic embryogenesis in tissue
culture is used to study the early stages of plant development. This helps in
understanding the molecular and genetic mechanisms that regulate embryogenesis.
Organogenesis: By inducing the formation of
specific organs (e.g., roots, shoots) from callus tissues, scientists can study
the hormonal and genetic controls of organ development.
3. Plant-Microbe Interactions:
- Symbiotic
Relationships: Tissue
culture systems are used to study symbiotic relationships, such as those
between plants and mycorrhizal fungi or nitrogen-fixing bacteria. This
helps in understanding how these interactions benefit plant growth and
development.
- Pathogenesis: Researchers use tissue
cultures to study plant responses to pathogenic microbes, identifying the
mechanisms of plant defense and susceptibility to diseases.
4. Genetics and Molecular Biology:
Gene Function and Expression:
o Genetic Transformation: Techniques such
as Agrobacterium-mediated transformation and biolistics allow for the
introduction of foreign genes into plant cells. This helps in studying gene
function by observing the phenotypic effects of overexpressing or silencing
specific genes.
o Reporter Genes: Reporter genes (e.g., GFP, GUS)
are used in tissue culture to monitor gene expression patterns. This helps in
understanding the spatial and temporal regulation of gene activity.
Genetic Mapping and Marker-Assisted Selection:
o Mapping Traits: Tissue culture is used to
produce large numbers of genetically identical plants, which are then used in
genetic mapping studies to identify the locations of genes associated with
desirable traits.
o Marker-Assisted Selection: Researchers use
tissue culture to propagate plants with specific genetic markers linked to
favorable traits. This accelerates the breeding process by allowing for the
selection of plants with desirable characteristics at the seedling stage.
CRISPR/Cas9 and Genome Editing:
o Targeted Mutations: CRISPR/Cas9
technology is applied in tissue culture to create precise genetic
modifications. This helps in studying gene function and developing plants with
improved traits, such as disease resistance or enhanced nutritional content.
5. Biochemistry:
Secondary Metabolite Production:
o Metabolic Engineering: Tissue cultures
are used to study and enhance the production of secondary metabolites, such as
alkaloids, flavonoids, and terpenoids. These compounds have pharmaceutical,
agricultural, and industrial applications.
o Pathway Elucidation: By manipulating
the culture conditions and genetic makeup of plant cells, researchers can
elucidate the biosynthetic pathways of secondary metabolites.
Enzyme Activity:
o In Vitro Assays: Tissue culture
provides a controlled environment for studying enzyme activity. Researchers can
isolate enzymes from cultured tissues and study their properties and functions
in detail.
o Metabolic Pathway Regulation: Understanding
how enzymes regulate metabolic pathways in response to different environmental
conditions and developmental stages is facilitated by tissue culture studies.
6. Environmental Stress Studies:
Abiotic Stress:
o Drought, Salinity, and Temperature: Tissue culture
systems allow researchers to simulate and study the effects of abiotic stresses
(e.g., drought, salinity, extreme temperatures) on plant cells. This helps in
identifying stress-responsive genes and pathways.
o Heavy Metal Toxicity: Researchers use
tissue cultures to study the mechanisms of heavy metal uptake, accumulation,
and detoxification in plants. This knowledge is crucial for developing plants
that can be used in phytoremediation.
Biotic Stress:
o Pathogen Resistance: Tissue cultures
are used to screen for and develop plant varieties with enhanced resistance to
pathogens, such as bacteria, fungi, and viruses. This involves studying the plant’s
immune responses at the cellular level.
o Herbivore Interaction: The interaction
between plants and herbivores can be studied in vitro to understand how plants
defend themselves against herbivory and how these interactions influence plant
secondary metabolite production.
Agriculture
Plant tissue
culture has revolutionized agriculture by providing methods for rapid plant
propagation, genetic improvement, and the production of disease-free plants.
Here’s a detailed look at its applications and impact on agriculture:
a. Micropropagation
Micropropagation is the practice of rapidly multiplying plant material
to produce a large number of progeny plants, using modern plant tissue culture
methods.
b. Production of Disease-Resistant Plants
Plant tissue
culture techniques are instrumental in developing and propagating
disease-resistant plant varieties. Plant tissue culture techniques are
essential for producing virus-free planting material, especially for
vegetatively propagated crops that are susceptible to viral infections.
c. Crop Improvement
Tissue culture
techniques accelerate plant breeding and the development of improved crop
varieties.
d. Germplasm Conservation
Tissue culture
plays a critical role in conserving plant genetic resources, particularly for endangered
species and crop varieties.
e. Phytoremediation
Tissue culture
techniques are used to produce plants that can clean up environmental
pollutants through phytoremediation.
- Hyperaccumulator
Plants:
- Selection
and Propagation: Identifying and propagating plants that can
accumulate high levels of heavy metals or other pollutants from soil and
water.
- Genetic Engineering:
- Enhanced
Phytoremediation: Introducing genes that enhance a
plant’s ability to detoxify and accumulate pollutants. Genetically
engineered plants can be more effective in cleaning up contaminated
environments.
Horticulture
Plant tissue culture has significantly
impacted horticulture by providing efficient methods for the propagation,
conservation, and improvement of ornamental plants, fruits, vegetables, and
other horticultural crops.
a.Ornamental Plants: Micropropagation is extensively used for ornamental plants such as
orchids, lilies, roses, and ferns. It ensures the uniformity and quality of
these high-value plants.
b.Fruits and Vegetables: Many fruit and vegetable crops, such as bananas,
strawberries, tomatoes, and potatoes, are propagated through tissue culture to
ensure uniformity, high yield, and disease resistance.
c.Genetic Improvement and Breeding
Plant tissue
culture techniques accelerate breeding programs and facilitate the genetic
improvement of horticultural crops.
Forestry
Plant tissue culture has become a
crucial tool in forestry for the propagation, conservation, and genetic
improvement of forest tree species. These techniques offer solutions to
challenges such as deforestation, forest degradation, and the need for
high-quality planting material.
a. Micropropagation in Forestry
Micropropagation
is a widely used tissue culture technique in forestry for the rapid
multiplication of forest tree species.
Applications:
- Timber
Trees: Species
such as teak (Tectona grandis), eucalyptus (Eucalyptus spp.), and poplar
(Populus spp.) are propagated through tissue culture to ensure uniformity
and high yield.
- Fruit
Trees: Forest
fruit trees like chestnut (Castanea spp.) and walnut (Juglans spp.) are
also propagated using these techniques.
- Multipurpose
Trees: Trees
that provide timber, fodder, and other resources, such as neem
(Azadirachta indica) and moringa (Moringa oleifera), benefit from tissue
culture propagation.
b. Conservation of Forest Genetic Resources
Tissue culture
techniques play a vital role in the conservation of forest genetic resources,
particularly for rare and endangered tree species.Involves storing plant
tissues at ultra-low temperatures in liquid nitrogen (-196°C) for long-term
conservation. Cryopreservation is suitable for conserving germplasm of rare and
endangered tree species.
c. Reforestation and Afforestation
Tissue culture
techniques provide high-quality planting material for reforestation and
afforestation projects.
·
Clonal
Forestry:
- Uniform
Plantations: Produces clonal plants with uniform growth
characteristics, which is important for establishing homogeneous forest
stands.
- Improved
Traits:
Clonal propagation ensures the consistent expression of desirable traits
such as fast growth, disease resistance, and high wood quality.
·
Rehabilitation
of Degraded Lands:
- Erosion
Control and Soil Improvement: Using tissue-cultured trees to
rehabilitate degraded lands, control erosion, and improve soil fertility.
- Species
Diversity:
Enhancing species diversity in reforestation projects by propagating a
wide range of tree species through tissue culture.
d. Production of Secondary Metabolites
Plant tissue
culture is used to produce valuable secondary metabolites from forest tree
species, which are compounds with pharmaceutical, agricultural, and industrial
applications.
Biotechnology
Plant tissue culture, a
cornerstone of plant biotechnology, involves the cultivation of plant cells,
tissues, or organs under sterile conditions on a nutrient culture medium. This
technique has revolutionized plant science and agriculture by enabling rapid
plant propagation, genetic improvement, and conservation.
Techniques of Plant Tissue Culture
Plant tissue
culture is a technique used to grow plants under sterile conditions on a
nutrient culture medium of known composition. This technique is used to produce
clones of a plant in a method known as micropropagation. Here’s a detailed
step-by-step overview of the process:
1. Selection of Plant Material
(Explant)
The selection of
plant material, or explant, is the first and crucial step in plant tissue
culture. The success of the entire process greatly depends on the type and
condition of the explant used. Here's a detailed breakdown of this step:
A. Choosing the Explant
Types of Explants:
- Meristematic
Tissues:
These are actively dividing tissues, usually found in the tips of shoots
and roots. They are often preferred due to their high regenerative
capacity and lower chances of contamination.
- Leaf Tissues: Sections
of leaves, often used for indirect organogenesis (callus formation
followed by shoot and root regeneration).
- Stem Tissues: Segments
of stems, including nodes and internodes, used for direct shoot
regeneration.
- Root Tissues: Less
commonly used but can be effective for certain species.
- Flower and
Fruit Tissues: Used for specific studies or species.
- Embryos and
Seeds:
Often used for the initial culture establishment and regeneration studies.
Criteria for Selecting
Explants:
- Species and
Variety:
Different species and varieties of plants may respond differently to
tissue culture conditions. Selection should be based on the purpose of the
culture (e.g., micropropagation, genetic studies).
- Health of
the Plant:
The source plant should be healthy, disease-free, and vigorously growing
to ensure the highest chances of success.
- Age of the
Tissue:
Younger tissues generally have a higher regenerative capacity and lower
levels of contamination.
B. Preparation of Explant
Collection: Collect the explant from the source plant using
clean, sterilized tools to minimize contamination.
Surface Sterilization:
- Rinsing: Rinse the
explant thoroughly under running tap water to remove surface dirt and
debris.
- Detergent
Wash:
Soak the explant in a mild detergent solution (e.g., Tween 20) for 10-15
minutes, followed by thorough rinsing with distilled water.
- Disinfection: Treat the
explant with a disinfectant solution (e.g., 70% ethanol for 30 seconds
followed by 2-10% sodium hypochlorite for 5-20 minutes). The concentration
and duration depend on the type of explant and the level of contamination
risk.
- Rinsing: Rinse the
disinfected explant multiple times with sterile distilled water to remove
any traces of disinfectant.
Size and Shape: Trim the explant to the appropriate size (usually
1-2 cm) under sterile conditions. The cut surfaces should be smooth to
facilitate better contact with the culture medium.
2. Preparation of Culture Medium
The culture medium
is a critical component in plant tissue culture, providing the necessary
nutrients and growth regulators for the explant to grow and develop. Commonly
used media include MS (Murashige and Skoog), B5 (Gamborg’s), and WPM (Woody
Plant Medium). Here's a detailed breakdown of how to prepare the culture medium:
A. Components of the Culture Medium
1. Macro and
Micronutrients
·
Macronutrients: These are required in larger quantities and include:
- Nitrogen
(N):
Provided as ammonium nitrate (NH₄NO₃) or potassium nitrate (KNO₃).
Nitrogen is essential for amino acid and protein synthesis.
- Phosphorus
(P):
Supplied as potassium dihydrogen phosphate (KH₂PO₄). It plays a key role
in energy transfer through ATP.
- Potassium
(K):
Added as potassium nitrate (KNO₃) or potassium sulfate (K₂SO₄). It is
important for enzyme activation and osmoregulation.
- Calcium
(Ca):
Usually provided as calcium chloride (CaCl₂) or calcium nitrate
(Ca(NO₃)₂). It is vital for cell wall stability and signaling.
- Magnesium
(Mg):
Supplied as magnesium sulfate (MgSO₄). It is a central component of the chlorophyll
molecule.
- Sulfur (S): Added as
magnesium sulfate (MgSO₄). It is a component of certain amino acids and
coenzymes.
·
Micronutrients: These are required in smaller quantities and
include:
- Iron (Fe): Provided
as ferrous sulfate (FeSO₄) or iron chelate (Fe-EDTA). It is essential for
chlorophyll synthesis and enzyme function.
- Manganese
(Mn):
Added as manganese sulfate (MnSO₄). It is important for photosynthesis
and enzyme activation.
- Zinc (Zn): Supplied
as zinc sulfate (ZnSO₄). It is necessary for enzyme function and protein
synthesis.
- Copper (Cu): Provided
as copper sulfate (CuSO₄). It is involved in photosynthesis and
respiration.
- Boron (B): Added as
boric acid (H₃BO₃). It plays a role in cell wall formation and membrane
integrity.
- Molybdenum
(Mo):
Supplied as sodium molybdate (Na₂MoO₄). It is a cofactor for nitrate
reductase enzyme.
2. Vitamins
- Commonly
used vitamins include:
- Thiamine
(Vitamin B1): Important for carbohydrate metabolism.
- Pyridoxine
(Vitamin B6): Involved in amino acid metabolism.
- Nicotinic
Acid (Niacin): Essential for respiration and lipid
metabolism.
3. Amino Acids: Amino acids such as glycine can be added to the medium
to provide a nitrogen source and stimulate growth.
4. Plant Growth
Regulators
- Auxins: Promote
root formation and callus induction (e.g., Indole-3-acetic acid (IAA),
Indole-3-butyric acid (IBA), Naphthalene acetic acid (NAA)).
- Cytokinins: Promote
shoot formation and cell division (e.g., 6-Benzylaminopurine (BAP),
Kinetin, Zeatin).
- Gibberellins: Promote
stem elongation and germination.
- Abscisic
Acid:
Regulates stress responses and inhibits growth.
5. Carbohydrates: Sucrose is the most commonly used carbohydrate source,
providing energy for growth and development.
6. Solidifying Agent:
Agar: A gelatinous substance derived from seaweed, used to
solidify the medium. It provides a support matrix for the explant.
B. Steps in Preparing the Culture Medium
1. Weighing and Mixing Components Accurately weigh the required amounts of
macronutrients, micronutrients, vitamins, amino acids, and plant growth
regulators. Dissolve the components in distilled water, usually starting with
macronutrients, followed by micronutrients, vitamins, and growth regulators.
2. Adjusting pH: Adjust
the pH of the medium to 5.7-5.8 using either 1N NaOH or 1N HCl. The pH is
crucial for nutrient availability and optimal growth.
3. Adding Carbohydrates: Add the required amount of sucrose (usually 20-30 g/L) to the solution.
4. Adding Agar: If a
solid medium is needed, add agar (usually 7-8 g/L) to the solution.
5. Sterilization
o Dispense the
prepared medium into culture vessels (e.g., flasks, jars, Petri dishes).
o Sterilize the
medium by autoclaving at 121°C and 15 psi for 15-20 minutes. This kills any
microbial contaminants.
6. Cooling and Solidifying: Allow the sterilized medium to cool and solidify under sterile
conditions. If using liquid medium, it can be used directly after cooling.
7. Storage: Store
the prepared culture medium at room temperature or in a refrigerator until use.
Ensure it is properly sealed to prevent contamination.
3. Inoculation
Inoculation
involves placing the sterilized explant onto the prepared culture medium under
sterile conditions. This step is critical to ensure that the explant has the
best chance of growth without contamination. Here’s a detailed breakdown of the
inoculation process:
A. Preparation for Inoculation
1. Sterile Environment
o Laminar Flow Hood: Work inside a
laminar flow hood or a clean bench to maintain a sterile environment. The hood
should be cleaned with a disinfectant (e.g., 70% ethanol) before starting the
procedure.
o Personal Preparation: Wear sterile
gloves, a lab coat, and a face mask to minimize contamination risks. Gloves
should be regularly wiped with ethanol during the procedure.
2. Sterilization of Tools
o Instruments: Sterilize all instruments
(e.g., forceps, scalpels, scissors) by autoclaving or by flaming with ethanol
and a Bunsen burner.
o Medium and Containers: Ensure that
culture vessels (e.g., Petri dishes, culture tubes, jars) containing the
sterilized medium are within easy reach and have been sterilized by
autoclaving.
B. Placing the Explant
1. Handling the Explant
o Transfer: Using sterilized forceps, carefully
transfer the sterilized explant to the culture vessel containing the prepared
culture medium.
o Placement: Position the explant so that it is in
firm contact with the surface of the culture medium. If using a solid medium,
ensure the explant is partially embedded or firmly resting on the medium.
2. Orientation
o Correct Orientation: Ensure the
correct orientation of the explant. For example, if using a stem segment, place
it horizontally or vertically depending on the protocol. Leaf explants should
be placed with the abaxial (lower) side in contact with the medium if this
promotes better regeneration.
3. Spacing
o Avoid Overcrowding: If inoculating
multiple explants in the same vessel, ensure they are adequately spaced to
allow for individual growth and to minimize competition and contamination
spread.
C. Sealing and Labeling
1. Sealing the Culture Vessel
o Preventing Contamination: Seal the
culture vessel with its lid, parafilm, or another suitable sealing material to
prevent contamination while allowing gas exchange. This is especially important
for vessels that need to maintain sterility over long periods.
2. Labeling
o Identification: Label each culture vessel with
relevant information such as the date of inoculation, type of explant, plant
species, and any specific treatment or medium composition.
o Tracking: Use waterproof markers or labels to
ensure that the information remains legible throughout the culture period.
4. Incubation
Incubation is a
crucial phase in plant tissue culture where the inoculated explants are placed
in a controlled environment to facilitate their growth and development. This
phase involves maintaining optimal conditions for temperature, light, humidity,
and other factors to ensure successful plant regeneration. Here’s a detailed
breakdown of the incubation process:
A. Environmental Conditions
1. Temperature
o Optimal Range: The optimal temperature for
most plant tissue cultures is between 25°C and 27°C. Some species may require
slightly different temperatures, so it’s important to adjust based on specific
requirements.
o Consistency: Maintain a constant temperature
to avoid stress on the explants. Fluctuations can adversely affect growth and
development.
2. Light
o Photoperiod: A common light/dark cycle is 16
hours of light and 8 hours of darkness. This mimics natural conditions and
supports photosynthesis.
o Light Intensity: Light intensity typically
ranges from 1000 to 3000 lux. This can vary depending on the plant species and
the stage of development. Fluorescent lamps are commonly used to provide
consistent and even lighting.
o Light Quality: Some cultures benefit from
specific light wavelengths (e.g., blue or red light). Specialized growth lights
can be used to enhance specific growth responses.
3. Humidity
o High Humidity: Maintain high humidity (about
60-80%) to prevent desiccation of the explants and to promote growth. This is
especially important for cultures in the early stages.
o Control: Use humidifiers or closed containers
to maintain consistent humidity levels.
4. Air Circulation
o Gas Exchange: Adequate air circulation is
essential for gas exchange. Culture vessels should be loosely capped or have
vents to allow the exchange of gases (CO₂ and O₂) while minimizing
contamination risks.
B. Observation and Monitoring
1. Regular Inspection
o Daily Checks: Inspect the cultures daily or
at regular intervals for signs of growth, contamination, and changes in the
explants. Use a sterile technique to handle and examine the cultures.
o Growth Indicators: Look for
indicators such as callus formation, shoot initiation, root development, and
overall health of the explant.
2. Contamination Control
o Identify Contamination: Contamination
can be bacterial, fungal, or algal. It often appears as cloudy media, unusual
colors, or fuzzy growths.
o Immediate Action: If
contamination is detected, remove the affected cultures immediately to prevent
it from spreading to other cultures.
3. Data Recording
o Documentation: Keep detailed records of
observations, including the date, type of growth, any signs of contamination,
and any interventions made. This helps in tracking progress and making
necessary adjustments.
5. Subculturing
Subculturing, also
known as reculturing or passage, is a vital step in plant tissue culture. It
involves transferring growing tissues or organs from one culture medium to
another to provide fresh nutrients, reduce the risk of contamination, and
promote further growth and development. Here's a detailed breakdown of the
subculturing process:
A. Purpose of Subculturing
1. Nutrient Renewal
o Nutrient Depletion: As explants
grow, they consume the nutrients in the culture medium. Subculturing to a fresh
medium replenishes essential nutrients and growth regulators.
o Waste Accumulation: Metabolic
byproducts can accumulate in the culture medium, potentially inhibiting growth.
Subculturing helps to reduce these waste products.
2. Space for Growth
o Overcrowding: As explants proliferate, they
may become overcrowded. Subculturing provides more space for individual
explants to grow.
o Physical Separation: Separating
shoots, roots, or callus can promote more uniform growth and reduce competition
for resources.
3. Phase Transition
o Induction to Differentiation: Subculturing
can be used to transition explants from one phase to another, such as from
callus induction to organogenesis or from shoot multiplication to root
induction.
B. Preparation for Subculturing
1. Sterile Environment
o Laminar Flow Hood: Conduct all
subculturing procedures under a laminar flow hood or sterile workspace to
maintain aseptic conditions.
o Personal Hygiene: Wear sterile
gloves, a lab coat, and a face mask. Disinfect gloves and instruments with
ethanol periodically.
2. Sterilization of Tools and Media
o Instruments: Sterilize all instruments (forceps,
scalpels, scissors) by autoclaving or by flaming with ethanol and a Bunsen
burner.
o Culture Vessels: Prepare and autoclave fresh
culture medium in sterile culture vessels (Petri dishes, jars, tubes).
C. Subculturing Procedure
1. Selection of Material
o Healthy Explants: Choose
healthy, actively growing tissues or organs for subculturing. Avoid any that
show signs of contamination or necrosis.
o Size and Quantity: Decide on the
appropriate size and number of explants to transfer based on the growth stage and
density.
2. Transferring Explants
o Handling Explants: Use sterilized
forceps and scalpels to carefully remove explants from the original culture
vessel.
o Cutting: If necessary, cut the explants into
smaller pieces. For example, divide callus into smaller sections or separate
individual shoots.
o Placement: Transfer the explants to the fresh
culture medium. Ensure they are properly positioned and in firm contact with
the medium. For shoots, partially embed the base in the medium; for callus,
spread evenly.
3. Sealing and Labeling
o Sealing: Seal the culture vessels with their
lids, parafilm, or other suitable materials to maintain sterility and humidity.
o Labeling: Label each culture vessel with
information such as the date of subculturing, type of explant, plant species,
and medium composition.
D. Incubation After Subculturing
1. Controlled Environment
o Growth Chamber: Place the subcultured vessels
in a growth chamber with controlled temperature, light, and humidity.
o Optimal Conditions: Maintain the
same optimal conditions as during initial incubation: temperature (25-27°C),
light intensity (1000-3000 lux), and photoperiod (16 hours light/8 hours dark).
2. Monitoring Growth
o Regular Observation: Monitor the
subcultured explants regularly for signs of growth, development, and any
contamination.
o Record Keeping: Document the progress of each
culture, noting any changes in growth patterns or issues that arise.
E. Frequency of Subculturing
1. Growth Rate
o Fast-Growing Cultures: Fast-growing
explants may need subculturing more frequently, every few weeks, to prevent
overcrowding and nutrient depletion.
o Slow-Growing Cultures: Slow-growing
explants may only require subculturing every few months.
2. Specific Requirements
o Species-Specific Needs: The frequency
and conditions for subculturing can vary widely among different plant species
and types of tissues being cultured. Tailor the subculturing schedule to meet
specific requirements.
6. Acclimatization
Acclimatization,
or hardening off, is the process of gradually adapting tissue-cultured
plantlets to the external environment. This step is crucial as plantlets grown
in vitro are often not equipped to handle the harsher conditions outside the
controlled laboratory environment. Here's a detailed breakdown of the
acclimatization process:
A. Purpose of Acclimatization
1.
Gradual
Transition
o Environmental Adjustment: Plantlets need
to transition from the high humidity, low light, and sterile conditions of in
vitro culture to the variable and less controlled conditions outside.
o Structural and Physiological Changes: In vitro
plantlets often have thin cuticles, underdeveloped root systems, and lack
mechanisms to handle stress. Acclimatization helps them develop these features.
B. Initial Acclimatization Phase
1. Controlled Environment
o High Humidity: Initially maintain high
humidity to prevent desiccation. This can be achieved using humidity domes,
plastic covers, or placing pots in a high-humidity chamber.
o Indirect Light: Expose plantlets to indirect
light to avoid stress from intense light. Gradually increase light exposure
over time.
2. Medium Transition
o Sterile Soil Mix: Transfer
plantlets to a sterile soil mix to provide better support and nutrients. The
mix can be a combination of peat, vermiculite, and perlite.
o Small Pots: Use small pots initially to allow easy
handling and monitoring of the plantlets.
3. Handling Plantlets
o Gentle Transfer: Gently remove the plantlets
from the culture vessels to avoid damaging delicate roots and shoots. Rinse off
any agar medium from the roots if necessary.
o Planting: Plant the plantlets in the prepared
pots, ensuring the roots are well covered and the shoots are above the soil
surface.
C. Intermediate Acclimatization Phase
1. Gradual Reduction in Humidity
o Ventilation: Gradually increase ventilation
by opening the humidity domes or plastic covers slightly each day. This helps
the plantlets adjust to lower humidity levels.
o Mist Spraying: Mist the plantlets regularly to
maintain moisture without keeping the environment too humid.
2. Increasing Light Intensity
o Natural Light: Gradually expose the plantlets
to more natural light. Start with shaded areas and slowly move them to brighter
locations.
o Artificial Light: If using
artificial light, increase the intensity and duration incrementally.
3. Nutrient Support
o Fertilization: Begin light fertilization with
a diluted balanced fertilizer to support growth. Avoid over-fertilizing, as
young plantlets are sensitive to nutrient levels.
D. Advanced Acclimatization Phase
1. Exposure to Outdoor Conditions
o Hardening Off Outdoors: Once plantlets
show strong growth and better-developed root systems, start exposing them to
outdoor conditions for a few hours each day.
o Gradual Increase: Gradually
increase the duration of outdoor exposure over several days to weeks. This
helps plantlets adjust to temperature variations, wind, and direct sunlight.
2. Monitoring and Care
o Watch for Stress: Monitor
plantlets closely for signs of stress, such as wilting, yellowing, or leaf
drop. Provide shade or bring them back indoors if needed.
o Watering: Adjust watering to ensure the soil
remains moist but not waterlogged. Over time, reduce the frequency of watering
to match outdoor conditions.
E. Final Transfer to Permanent Location
1. Selection of Planting Site
o Appropriate Environment: Choose a
planting site that matches the needs of the plant species. Consider factors
such as sunlight, soil type, and drainage.
o Preparation: Prepare the planting site by
ensuring the soil is well-draining and enriched with organic matter if
necessary.
2. Transplanting
o Timing: Transplant plantlets during a time of
day when conditions are mild, such as early morning or late afternoon, to
reduce transplant shock.
o Planting Depth: Plant at the same depth as they
were in the pots to ensure stability and promote root growth.
3. Post-Transplant Care
o Watering: Water the transplanted plantlets
thoroughly after planting to settle the soil around the roots.
o Mulching: Apply mulch around the base of the
plantlets to conserve moisture and reduce weed competition.
4. Ongoing Care
o Regular Monitoring: Continue to
monitor the plantlets for growth and health. Provide additional support such as
staking if necessary.
o Pest and Disease Management: Implement pest and disease management practices to protect the young plants.
Applications of Plant Tissue Culture
Plant tissue
culture is a technique used to grow plant cells, tissues, or organs under
sterile conditions on a nutrient culture medium. This method has a wide range
of applications in research, agriculture, and horticulture. Here are some
detailed applications of plant tissue culture, along with examples:
1. Micropropagation
Micropropagation is the rapid multiplication of plant material to
produce a large number of progeny plants, using modern tissue culture methods.
- Example: Orchids
and bananas are often propagated through tissue culture to ensure
uniformity and disease-free plants. The process involves taking small
tissue samples (explants) from the parent plant and growing them in vitro
under controlled conditions.
2. Genetic Engineering
Plant tissue culture is
a key tool in genetic engineering, enabling the introduction of new genes into plant cells via
techniques such as Agrobacterium-mediated transformation or biolistics (gene
gun),CRISPR/Cas9
(Precise editing of plant genomes to improve traits like disease resistance,
drought tolerance, and crop yield).
- Example: Bt
Cotton is a genetically modified cotton variety that contains a
gene from the bacterium Bacillus thuringiensis (Bt), which
provides resistance to certain pests. The gene is introduced into cotton
cells through tissue culture techniques.
3. Production of Secondary Metabolites
Certain plants
produce valuable secondary metabolites, such as alkaloids, flavonoids, and
terpenoids, which have pharmaceutical and industrial applications. Tissue
culture techniques can be used to produce these compounds in vitro.
- Example: Shikonin
is a compound produced by the plant Lithospermum erythrorhizon
and has applications in cosmetics and medicine. Tissue culture methods can
be used to produce shikonin on a large scale.
4. Germplasm Conservation
Tissue culture
techniques are used for the long-term storage of plant genetic resources. This
is particularly important for the conservation of endangered species and the
preservation of genetic diversity.
- Example: Cryopreservation
involves storing plant tissues at ultra-low temperatures (usually in
liquid nitrogen) to preserve genetic material for future use. Many
endangered plant species, such as certain orchids, are conserved using
this method.
5. Somatic Hybridization
Somatic hybridization
involves the fusion of protoplasts (plant cells without cell walls) from
different species or varieties to create hybrid plants with desirable traits.
- Example: Pomato
is a hybrid between a potato (Solanum tuberosum) and a tomato (Solanum
lycopersicum). Protoplast fusion techniques can be used to combine
the genetic material of both plants, resulting in a plant that produces
both potatoes and tomatoes.
6. Virus Elimination
Plant tissue
culture can be used to produce virus-free plants through a process called
meristem culture. This is particularly important for plants that are
vegetatively propagated and prone to viral infections.
- Example: Potato
plants are often propagated through tissue culture to eliminate viruses
and ensure healthy planting material.
7. Somaclonal Variation
Tissue culture can
induce genetic variations known as somaclonal variations. These variations can
be used to select plants with desirable traits, such as disease resistance or
improved yield.
- Example: Sugarcane
breeding programs utilize somaclonal variation to develop new varieties
with improved characteristics.
8. Embryo Rescue
Embryo rescue is a
technique used to save embryos from hybrid crosses that would otherwise fail to
develop in vivo. This is useful in plant breeding, especially when crossing
distantly related species.
- Example: Interspecific
hybrids in citrus breeding often require embryo rescue to obtain
viable plants from crosses between different Citrus species.
9. Synthetic Seeds
Synthetic seeds
are artificially encapsulated somatic embryos or other tissues that can be used
for sowing as a seed. This technology aids in the conservation and easy
distribution of plant genetic material.
- Example: Synthetic
seeds of medicinal plants such as Bacopa monnieri
(Brahmi) are developed for easier storage and propagation.
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