Unit 4: In vitro propagation - Subjective Questions

Callus is an unorganized, undifferentiated mass of actively dividing parenchymatous cells that develops from an explant when cultured on a suitable nutrient medium under aseptic conditions.

Significance in plant tissue culture:

• Acts as a source of totipotent cells capable of regenerating whole plants.
• Serves as starting material for organogenesis (shoot/root formation) and somatic embryogenesis.
• Used to establish cell suspension cultures for secondary metabolite production.
• Facilitates genetic transformation and production of transgenic plants.
• Enables studies on cellular differentiation, biochemistry, and mutation.
• Useful for micropropagation and production of virus-free plants.

The formation of callus is generally induced by an appropriate balance of auxins and cytokinins in the culture medium.

The preparation of callus involves the following systematic steps:

1. Selection of explant: A healthy, actively growing tissue (leaf, stem, root, cotyledon, etc.) is chosen from a disease-free mother plant.
2. Washing: The explant is washed under running tap water to remove dust and surface debris.
3. Surface sterilization: The explant is treated with sterilants such as 70% ethanol (30 sec) followed by 0.1% mercuric chloride () or sodium hypochlorite for a few minutes, then rinsed with sterile distilled water 3–4 times.
4. Excision: Under a laminar air flow chamber, the sterilized explant is cut into small pieces (about 0.5–1 cm) using sterile instruments.
5. Inoculation: The explant pieces are placed onto a solid nutrient medium (e.g., MS medium) supplemented with growth regulators (auxins + cytokinins).
6. Incubation: Cultures are kept in a growth room at with appropriate light/dark cycles.
7. Callus induction: Within 2–4 weeks, a proliferating mass of callus develops at the cut ends of the explant.
8. Subculturing: Callus is periodically transferred to fresh medium every 3–4 weeks to maintain growth.

An explant is a piece of living plant tissue or organ that is excised from a donor plant and cultured in vitro to initiate a culture such as callus.

Various types of explants used for callus induction:

• Leaf explants – lamina, petiole segments
• Stem/internodal explants – nodal and internodal segments
• Root explants – root tips and segments
• Meristematic tissues – shoot tips, apical meristems
• Cotyledons and hypocotyls – common in dicots
• Embryos – mature and immature
• Endosperm – for triploid callus
• Anther/pollen (microspore) – for haploid callus
• Floral parts – ovary, petals, sepals

The choice of explant depends on the plant species, purpose of the culture, and regeneration potential of the tissue. Younger, actively dividing tissues generally show better callusing response.

Plant growth regulators (PGRs) are essential for inducing and maintaining callus. The auxin-to-cytokinin ratio is the key determinant of the developmental response.

Role of Auxins:

• Promote cell division and cell elongation.
• Induce callus initiation and proliferation.
• Common auxins: 2,4-D (2,4-dichlorophenoxyacetic acid), NAA, IAA, IBA.
• 2,4-D is the most potent for callus induction.

Role of Cytokinins:

• Promote cell division (cytokinesis) and shoot differentiation.
• Common cytokinins: BAP (6-benzylaminopurine), Kinetin, Zeatin, 2-iP.

Effect of ratio:

• High auxin : low cytokinin → root formation.
• Low auxin : high cytokinin → shoot formation.
• Balanced/intermediate ratio → callus proliferation (undifferentiated growth).

This concept was established by Skoog and Miller (1957), forming the basis of controlling in vitro morphogenesis.

Surface sterilization is a critical step that removes microbial contaminants (bacteria, fungi, spores) from the surface of the explant without damaging the plant tissue.

Importance:

• Prevents microbial contamination of the culture medium which is rich in nutrients.
• Ensures aseptic culture conditions essential for callus growth.
• Increases the survival rate and success of cultures.
• Avoids competition between microbes and plant tissue for nutrients.

Commonly used sterilants:

• Ethanol (70%) – 30 seconds to 1 minute
• Mercuric chloride (, 0.1%) – 2–10 minutes
• Sodium hypochlorite (NaOCl, 1–2%) – 5–15 minutes
• Hydrogen peroxide, calcium hypochlorite – alternatives

Precautions:

• Optimal concentration and exposure time must be chosen to avoid tissue toxicity.
• Sterilant must be thoroughly rinsed off with sterile distilled water (3–4 times).
• A surfactant (Tween-20) may be added to improve wetting.

MS medium, developed by Murashige and Skoog (1962), is the most widely used nutrient medium for plant tissue culture due to its high salt content.

Major components:

• Macronutrients: Nitrogen (as and ), Phosphorus, Potassium, Calcium (), Magnesium ().
• Micronutrients: Iron (as Fe-EDTA), Manganese, Zinc, Boron, Copper, Molybdenum, Cobalt.
• Vitamins: Thiamine (), Nicotinic acid, Pyridoxine (), Myo-inositol.
• Carbon source: Sucrose (3%, i.e., 30 g/L) as energy source.
• Amino acid: Glycine.
• Growth regulators: Auxins and cytokinins as required.
• Gelling agent: Agar (0.6–0.8%) for solid medium.

Other parameters:

• pH adjusted to 5.6–5.8 before autoclaving.
• Sterilized by autoclaving at , 15 psi for 15–20 minutes.

The high concentration of inorganic salts makes MS medium suitable for callus induction and rapid growth of many plant species.

Callus can be classified based on its texture and appearance:

Key point: Friable callus is preferred for establishing single-cell suspension cultures because it disperses readily in liquid medium, whereas compact callus is more suitable for differentiation and regeneration studies.

Totipotency is the inherent ability of a single plant cell to divide, differentiate, and regenerate into a complete, whole plant when provided with appropriate nutritional and hormonal conditions.

Concept: The term was coined based on the work of Haberlandt (1902), and demonstrated practically by Steward using carrot cells.

Relevance to callus culture:

• Each cell of the callus retains the complete genetic information of the parent plant.
• These totipotent cells can be induced to undergo organogenesis (shoot/root formation) or somatic embryogenesis.
• Enables regeneration of whole plants from callus, forming the basis of micropropagation.
• Fundamental to genetic engineering, as transformed single cells can regenerate into transgenic plants.

Significance: Totipotency is the foundational principle behind all plant tissue culture techniques, allowing mass clonal propagation and germplasm conservation.

The growth of callus in culture follows a characteristic sigmoid (S-shaped) growth curve consisting of the following phases:

1. Lag Phase:

   • Initial phase where cells prepare for division.
   • Little or no increase in cell number.
   • Cells adapt to the new medium and synthesize necessary metabolites.

2. Log (Exponential) Phase:

   • Rapid cell division and proliferation.
   • Maximum rate of increase in cell number and biomass.

3. Linear Phase:

   • Cell division slows but cell expansion increases.
   • Growth continues at a constant rate.

4. Deceleration (Stationary) Phase:

   • Growth rate declines due to nutrient depletion and accumulation of toxic metabolites.
   • Cell division and expansion slow down and stop.

5. Death/Decline Phase:

   • If not subcultured, cells begin to die due to exhaustion of nutrients.

Note: To maintain viability, the callus must be subcultured onto fresh medium (usually during the late log or linear phase) at regular intervals of 3–4 weeks.

Subculturing is the process of transferring a portion of the growing callus onto fresh nutrient medium at regular intervals to sustain its growth and viability.

Reasons why subculturing is necessary:

• Nutrient depletion: The original medium becomes exhausted of essential nutrients over time.
• Accumulation of toxic metabolites: Waste products and phenolic compounds accumulate and inhibit growth.
• Drying of medium: The medium loses moisture, becoming unsuitable for growth.
• Maintenance of growth: Fresh medium re-initiates active cell division.
• Prevention of browning/death: Removes necrotic tissue and phenolic exudates.
• Mass multiplication: Allows continuous increase in callus quantity.

Procedure: Under aseptic conditions in a laminar air flow chamber, a small healthy piece of callus is transferred to freshly prepared medium every 3–4 weeks.

Note: Excessive subculturing may lead to somaclonal variation and loss of regeneration potential.

Several factors influence the induction and proliferation of callus:

1. Explant factors:

• Type of explant (leaf, stem, root, etc.)
• Age and physiological state – younger tissues respond better.
• Position on donor plant and genotype.

2. Medium composition:

• Nutrients (macro and micronutrients).
• Carbon source (sucrose concentration).
• Vitamins and organic supplements.

3. Plant growth regulators:

• Auxin : cytokinin ratio is critical for callus induction.

4. Physical factors:

• Temperature ( optimal).
• Light/dark conditions – callus often initiated in dark.
• pH of medium (5.6–5.8).
• Humidity.

5. Aseptic conditions:

• Proper sterilization to prevent contamination.

6. Gaseous environment:

• Availability of oxygen and within culture vessels.

Optimizing these factors ensures maximum callus induction frequency and healthy growth.

Browning (or blackening) of callus is a common problem in tissue culture caused by the oxidation of phenolic compounds released from wounded/cut plant tissues. These oxidized phenols are toxic and can inhibit growth or kill the explant.

Causes:

• Release of phenolic compounds upon tissue cutting.
• Action of enzymes like polyphenol oxidase (PPO) and peroxidase.
• Common in woody plants and tannin-rich species.

Measures to control browning:

• Adding antioxidants to the medium – e.g., ascorbic acid, citric acid.
• Using activated charcoal (0.2–3%) to absorb phenolics.
• Frequent subculturing to fresh medium.
• Adding polyvinylpyrrolidone (PVP) to adsorb phenols.
• Incubating cultures in the dark initially.
• Reducing tissue injury during excision.
• Soaking explants in antioxidant solution before inoculation.
• Maintaining cultures at lower temperature.

Controlling browning is essential for successful establishment and survival of callus cultures.

Both are types of in vitro culture systems but differ in nature and application:

Relationship: Cell suspension cultures are established by transferring friable callus into a liquid medium and agitating it on a rotary/orbital shaker (100–150 rpm), allowing cells to disperse.

Callus induction from leaf explant involves the following steps:

1. Selection: Young, healthy, fully expanded leaves are collected from a disease-free plant.
2. Washing: Leaves are washed thoroughly under running tap water with a mild detergent, then rinsed.
3. Surface sterilization (under laminar air flow):
   • Dip in 70% ethanol for 30 seconds.
   • Treat with 0.1% for 3–5 minutes.
   • Rinse 3–4 times with sterile distilled water.
4. Excision: The leaf is cut into small segments (about cm), ensuring the midrib/veins are included as they respond well.
5. Inoculation: Segments are placed with the abaxial (lower) surface in contact with MS medium supplemented with 2,4-D + BAP/Kinetin.
6. Incubation: Cultures are kept at , often in dark for the first few days.
7. Callus formation: Callus proliferates from the cut edges and veins within 2–3 weeks.
8. Subculture: Transfer to fresh medium every 3–4 weeks.

Leaf explants are widely used because they are abundant, easy to sterilize, and highly responsive.

Callus culture has numerous applications in plant biotechnology:

• Micropropagation: Rapid clonal multiplication of elite plants via regeneration.
• Somatic embryogenesis & organogenesis: Production of plantlets.
• Production of secondary metabolites: Alkaloids, flavonoids, pigments, drugs (via suspension cultures derived from callus).
• Genetic transformation: Callus used as target tissue for gene transfer to develop transgenic plants.
• Somaclonal variation: Generation of genetic variability for crop improvement.
• Production of haploid/homozygous plants: From anther-derived callus.
• Germplasm conservation: Storage of valuable plant material.
• Synthetic seed production: Using somatic embryos from callus.
• Mutation studies and selection of desirable traits (e.g., disease/salt resistance).
• Protoplast isolation for somatic hybridization.
• Basic research on cell differentiation and metabolism.

Thus, callus culture is a cornerstone technique in modern plant biotechnology.

These two processes are central to plant tissue culture and morphogenesis:

Dedifferentiation:

• It is the process by which mature, differentiated cells revert back to a meristematic (undifferentiated) state and regain the ability to divide.
• When an explant is placed on medium with appropriate growth regulators, its differentiated cells dedifferentiate to form callus.

Redifferentiation:

• It is the ability of dedifferentiated (callus) cells to differentiate again into organized structures such as shoots, roots, or embryos.
• This is achieved by manipulating the auxin : cytokinin ratio in the medium.

Sequence in tissue culture:

Significance: The interplay of dedifferentiation and redifferentiation demonstrates cellular totipotency and is the basis for regeneration of whole plants from callus.

Essential equipment:

• Laminar Air Flow (LAF) chamber: Provides sterile working area with HEPA-filtered air.
• Autoclave: For sterilizing media, glassware, and instruments at , 15 psi.
• pH meter: To adjust medium pH (5.6–5.8).
• Weighing balance: For accurate measurement of chemicals.
• Hot air oven: For dry sterilization of glassware.
• Growth/culture room: Maintains controlled temperature, light, and humidity.
• Refrigerator/freezer: For storing stock solutions and chemicals.
• Magnetic stirrer, water bath, distillation unit.
• Glassware: Culture tubes, flasks, Petri dishes, beakers.
• Instruments: Forceps, scalpels, blades, spirit lamp.

Aseptic techniques:

• Surface sterilization of explants.
• Working inside the LAF chamber, wiping with 70% ethanol.
• Flame-sterilizing instruments frequently.
• Sterilizing media and glassware by autoclaving.
• Using UV light in the LAF before use.
• Wearing clean lab coats and gloves.

Strict maintenance of asepsis prevents microbial contamination, which is the most common cause of culture failure.

Somaclonal variation refers to the genetic and phenotypic variation observed among plants regenerated from somatic cells cultured in vitro (such as callus or cell cultures).

How it arises:

• Chromosomal changes: Aneuploidy, polyploidy, deletions, translocations, inversions.
• Gene mutations: Point mutations in DNA.
• Epigenetic changes: DNA methylation, activation of transposable elements.
• Prolonged culture and repeated subculturing increase the frequency of variation.
• High concentration of growth regulators (especially 2,4-D) can be mutagenic.

Significance / Applications:

• Source of genetic variability for crop improvement.
• Selection of variants with disease resistance, salt/drought tolerance, higher yield.
• Useful when natural variation is limited.

Disadvantage:

• Undesirable in micropropagation where genetic uniformity (true-to-type clones) is required.

Thus, somaclonal variation can be both a useful tool and an unwanted problem depending on the objective.

The classic concept proposed by Skoog and Miller (1957) using tobacco pith tissue established that the ratio of auxin to cytokinin, rather than their absolute concentrations, controls the pattern of organ differentiation in callus.

Effect of different ratios:

• High auxin : Low cytokinin

  • Promotes root formation (rhizogenesis).

• Low auxin : High cytokinin

  • Promotes shoot formation (caulogenesis).

• Intermediate / Balanced ratio (auxin ≈ cytokinin)

  • Promotes callus proliferation (undifferentiated growth).

Represented as:

Significance:

• Forms the fundamental basis for controlled morphogenesis in vitro.
• Enables scientists to direct differentiation towards callus, roots, or shoots as desired.
• Essential for micropropagation and plant regeneration protocols.

This principle remains a cornerstone of plant tissue culture.

Callus induction from stem/internodal explant involves the following steps:

1. Selection: Young, tender stem segments are collected from a healthy actively growing plant.
2. Washing: Segments are washed under running tap water, followed by a dilute detergent wash and rinsing.
3. Surface sterilization (in laminar air flow):
   • Treat with 70% ethanol for 30 seconds.
   • Immerse in 0.1% mercuric chloride () for 3–5 minutes.
   • Rinse 3–4 times with sterile distilled water.
4. Excision: Cut the sterilized stem into internodal segments (0.5–1 cm), removing the nodes.
5. Inoculation: Place the segments horizontally on MS medium supplemented with auxins (2,4-D/NAA) and cytokinins (BAP/Kinetin).
6. Incubation: Maintain at under controlled light/dark conditions.
7. Callus formation: Callus develops from the cut ends and cambial region within 2–4 weeks.
8. Subculturing: Transfer callus to fresh medium every 3–4 weeks.

Advantage: Internodal explants contain actively dividing cambial cells, giving a good callusing response.