FTIR- Fourier Transform Infrared Spectroscopy

Principle, Methods, Applications, Tools, and Chemical Identification

1. Principle of FTIR

Fourier Transform Infrared Spectroscopy (FTIR) is an analytical technique used to identify organic, polymeric, and, in some cases, inorganic materials. The principle is based on the absorption of infrared radiation by a sample, resulting in vibrational transitions in molecular bonds.

• IR Radiation is passed through a sample.
• Molecules absorb specific frequencies of IR light that match their natural vibrational frequencies.
• The absorption pattern (spectra) is like a molecular fingerprint.
• The interferogram is converted into a spectrum using a Fourier Transform algorithm.

2. Methods of FTIR

There are two main modes of FTIR analysis:

a. Transmission Mode

• Sample is placed directly in the path of the IR beam.
• Common for liquids and transparent solids.

b. Attenuated Total Reflectance (ATR)

• IR light reflects internally through a crystal in contact with the sample.
• Ideal for solids, powders, and films without preparation.

c. Diffuse Reflectance (DRIFTS)

• Suitable for powdered or rough-surfaced samples.
• IR beam is reflected and scattered by the sample.

d. Reflectance/Specular Reflectance

• Used for thin films or surface coatings.

3. Applications of FTIR

FTIR is widely applied in several fields:

• Pharmaceuticals: Identifying functional groups in drug compounds.
• Polymers: Analyzing polymer structures and degradation.
• Environmental Science: Detecting pollutants or contaminants.
• Food Industry: Identifying food adulterants or quality testing.
• Nanotechnology: Monitoring surface modifications and nanoparticle functionalization.
• Botany and Biotech: Identifying phytochemicals and metabolites in plant extracts or callus culture.

4. Tools and Components Used in FTIR

• IR Source: Typically, a Globar (silicon carbide) or Nernst glower.
• Interferometer: Michelson interferometer to modulate the IR signal.
• Beam Splitter: Divides the IR beam (commonly made of KBr or CaF₂).
• Sample Holder/Stage: Holds solid, liquid, or powdered samples.
• Detector: Converts IR signal to an electrical signal (DTGS or MCT detectors).
• Computer + Software: For Fourier transform and spectrum analysis.

5. Common Chemical Names Detected in FTIR

FTIR identifies functional groups by detecting bond vibrations. Examples:

Functional Group	Wavenumber (cm⁻¹)	Typical Compounds Detected
–OH (hydroxyl)	3200–3600	Alcohols, Phenols
–NH (amine)	3300–3500	Amines, Proteins
C=O (carbonyl)	1650–1750	Aldehydes, Ketones, Esters
–CH (alkane/alkene)	2800–3100	Hydrocarbons
–COOH (carboxylic)	1700–1725 & 2500–3300	Carboxylic acids
–C≡C, –C≡N	2100–2260	Alkynes, Nitriles
–C–O–C– (ether)	1000–1300	Ethers, Glycosides

These are often used to identify:

• Flavonoids
• Phenolics
• Alkaloids
• Terpenoids
• Carbohydrates
• Proteins/Peptides

FTIR Procedure

To launch the FTIR program, double-click the OMNIC icon.

⬇️
From the Collect pull-down menu, select Setup, and select the following:

• No. of scans = 8
• Resolution = 2 cm⁻¹
• Zero filling = None
• Final format = Absorbance

⬇️
From the Edit pull-down menu, select Options. Click on the Collect tab at the top. Click the “Collect to New Window” box to deselect it. Click OK to close the window.

⬇️
Select Background from the Collect pull-down menu (Repeat background collection every 30 minutes to 1 hr. if the humidity is high).

⬇️
From the Collect pull-down menu, select Collect. Sample: Place the sample in the holder in the IR, then select Yes. Add to window = Yes

⬇️
From the Process pull-down menu, select: Automatic Baseline. Correct old spectrum shown in red and the new spectrum shown in blue. Click on the red one. From the Edit pull-down select Clear from the Process pull-down menu, select % Transmittance.

⬇️
From the View pull-down menu, select Display Limits (to change the axes of the graph).

⬇️
During spectrum display, click on T (for text) at the bottom of the page. Click on peaks of interest to label.

⬇️
From the File pull-down menu, select Print.

⬇️
To Run the Next Sample: from the Window pull-down menu, select New Window (more than 7 open windows will crash OMNIC).

⬇️
Repeat steps 4–8 above.

KBr Pellet Procedure for Solid Samples

• Take about 1/8th of the solid sample on a micro spatula and about 0.25–0.50 teaspoons of KBr.
• Mix thoroughly in a mortar while grinding with the pestle. If the sample is in large crystals, grind the sample separately before adding KBr.
• Place just enough spl. to cover the bottom in the pellet die.
• Place in press and press at 5000–10000 psi.
• Carefully remove the pressed sample from the die and place it in the FTIR sample holder.
• The pressed disc should be nearly clear if properly made. If it is translucent, regrind and repress.

 

 

Callus Culture: A Comprehensive Overview of Induction, Sub culturing, Classification, and Biotechnological Applications

Callus Culture

Callus culture is a fundamental technique in plant tissue culture, involving the growth of undifferentiated plant cell masses in a controlled, artificial environment. Here's a more in-depth look:

What is Callus?

A callus is an unorganized, proliferating mass of parenchyma cells. It forms when plant tissues are subjected to certain stimuli, particularly in response to wounding or when exposed to specific plant growth regulators. In essence, it's a mass of plant cells that have "dedifferentiated," meaning they've lost their specialized functions and reverted to a more basic, dividing state.

The Callus Culture Process:

1. Explant Selection:
• The process begins with selecting a suitable plant tissue, known as an explant. This can be a piece of leaf, stem, root, or other plant part.
2. Sterilization:
• Strict sterilization is crucial to prevent contamination by microorganisms. The explant is thoroughly sterilized to eliminate any bacteria or fungi.
3. Culture Medium:
• The explant is placed on a nutrient-rich culture medium. This medium typically contains:

• Essential mineral salts.
• Vitamins.
• Sugars (as an energy source).
• Plant growth regulators (hormones), such as auxins and cytokinins, which are critical for inducing callus formation.

4. Incubation:
• The cultures are incubated under controlled environmental conditions, including temperature, light, and humidity.
5. Callus Formation:
• Over time, the explant cells begin to divide and form a callus.
6. Subculturing:
• To maintain the callus culture, portions of the callus are periodically transferred to fresh culture medium (subcultured). This provides a continuous supply of nutrients and prevents the accumulation of waste products.
7. Regeneration (Optional):
• Depending on the desired outcome, the callus can be induced to regenerate whole plants through:

• Organogenesis: The formation of organs (shoots and roots) from the callus 
• Somatic embryogenesis: The formation of embryos from the callus cells.

Applications of Callus Culture:

• Micropropagation: Rapidly producing large numbers of genetically identical plants.
• Genetic engineering: Introducing foreign genes into plant cells.
• Production of secondary metabolites: Obtaining valuable compounds (e.g., pharmaceuticals) from plant cells.
• Plant breeding: Creating new plant varieties.
• Germplasm preservation: Conserving rare or endangered plant species.

Subculture of Callus

Subculturing refers to the transfer of callus from an old medium to a fresh medium after a certain time (typically 3–4 weeks), to maintain active growth and prevent browning or senescence. It helps in:

Purpose of Subculturing:

• Replenishing Nutrients:
• As callus grows, it depletes the nutrients in the culture medium. Subculturing involves transferring the callus to a fresh medium, ensuring a continuous supply of essential nutrients for healthy growth.
• Preventing Accumulation of Waste Products:
• Metabolic byproducts can accumulate in the culture medium, potentially inhibiting callus growth or causing toxicity. Subculturing helps to remove these waste products.
• Maintaining Callus Viability:
• Regular subculturing helps to maintain the viability and vigor of the callus culture, preventing it from senescence or death.
• Promoting Continued Growth:
• By providing fresh medium, subculturing stimulates continued cell division and proliferation, ensuring a consistent supply of callus tissue.
• Controlling Callus Characteristics:
• Sub culturing can also be used to influence the characteristics of the callus, for example by changing the hormone composition of the media.

The Sub culturing Process:

1. Preparation:

• A fresh batch of sterile culture medium is prepared.
• Sterile tools and a laminar flow hood are used to maintain aseptic conditions.

2. Callus Transfer:

•  A portion of the actively growing callus is carefully excised from the existing culture.
• This callus tissue is then transferred to the fresh culture medium.

3. Incubation:
• The subcultured callus is incubated under controlled environmental conditions, such as temperature, light, and humidity.
4. Frequency:
• The frequency of subculturing depends on the growth rate of the callus and the specific requirements of the plant species.

• Maintaining the viability of callus 
• Enhancing biomass
• Inducing differentiation (if required)
• Avoiding nutrient depletion and accumulation of toxic metabolites

Types of Callus Based on Texture and Color

1. Friable Callus

In plant tissue culture, "friable callus" refers to a specific texture of callus tissue. Here's a breakdown:

Callus:

A callus is an undifferentiated mass of plant cells. It forms in response to wounding or when plant tissue (an explant) is placed on a culture medium containing plant growth regulators.

Friable Callus:

• This type of callus is characterized by its loose, crumbly, and easily separable texture. The cells are loosely attached and have a relatively high-water content.
•  In contrast to a "compact callus," which is dense and firm, a friable callus is soft and delicate.
•  Friable callus is often desirable for certain applications in plant biotechnology, particularly for:
• Cell suspension cultures: Friable callus can be easily broken up and transferred to liquid media for cell suspension cultures, which are used for producing secondary metabolites or for genetic transformation.
• Somatic embryogenesis: Some friable callus types are more conducive to the development of somatic embryos, which can then be grown into whole plants.
• Loose, crumbly, and soft texture
• Useful for suspension culture
• Usually light in color (whitish or yellowish)

2. Compact Callus

When discussing callus culture in plant tissue culture, "compact callus" refers to a specific type of callus with distinct characteristics. Here's a breakdown:

Characteristics of Compact Callus:

• Dense and Firm:
• Unlike friable callus, which is loose and crumbly, compact callus is characterized by its tightly packed cells, resulting in a firm and dense texture.
• Tightly Aggregated Cells:
• The cells within a compact callus are closely adhered to one another, contributing to its solid appearance.
• Often Green:
• Compact callus may often have a greenish coloration, especially if it's derived from tissues with chloroplasts.
• Relatively Dry:
• Compared to a friable callus, a compact callus will usually contain less water.

Contrasting with Friable Callus:

• It's important to differentiate compact callus from friable callus. Friable callus has a loose, crumbly texture, making it more suitable for cell suspension cultures.
• Compact callus is denser.
• The texture of the callus is very dependent on the hormone balances within the growth medium.

Factors Influencing Callus Type:

• Plant Growth Regulators:
• The ratio of auxins to cytokinins in the culture medium significantly influences callus texture. Different ratios can favor the formation of either compact or friable callus.
• Plant Species and Genotype:
• Different plant species and even different cultivars within a species exhibit variations in callus texture.
• Culture Conditions:
• Environmental factors like light, temperature, and the composition of the basal medium also play a role.

Significance:

•  While friable callus is often preferred for cell suspension cultures, compact callus can be suitable for other applications in plant tissue culture.
• It is a form of callus that is produced, and then can be manipulated to produce different plant parts, through the changing of the hormone balances in the growth medium.
• Hard, dense, and tightly packed cells
• Often greenish or cream-colored
•  Less suitable for suspension cultures

3. Embryogenic Callus

Embryogenic callus is a specialized form of callus that holds significant importance in plant tissue culture. Here's a detailed explanation:

Key Characteristics:

• Potential for Somatic Embryogenesis:
• The defining characteristic of embryogenic callus is its ability to give rise to somatic embryos. Somatic embryos are embryos that develop from plant cells other than zygotes (fertilized eggs).
• Organized Structure:
• Unlike undifferentiated callus, embryogenic callus often exhibits a degree of organization, with cells that are predisposed to develop into embryos.
• Distinct Cellular Features:
• Embryogenic cells tend to be small, densely cytoplasmic, and have prominent nuclei.
• High Regeneration Capacity:
• This type of callus has a high capacity for regeneration, meaning it can efficiently produce whole plants.

Significance in Plant Tissue Culture:

• Efficient Plant Regeneration:
• Embryogenic callus is highly valuable for regenerating large numbers of plants, especially in species that are difficult to propagate through conventional methods.
• Genetic Transformation:
• It serves as an excellent target for genetic transformation techniques, allowing for the introduction of desired genes into plants. The resulting somatic embryos can then develop into transgenic plants.
• Clonal Propagation:
• Embryogenic callus allows for the clonal propagation of plants, ensuring that all regenerated plants are genetically identical to the parent plant.
• Plant Breeding:
• It's utilized in plant breeding programs to generate new plant varieties.

Factors Influencing Embryogenic Callus Formation:

• Plant Growth Regulators:
• The type and concentration of plant growth regulators, particularly auxins, play a crucial role in inducing embryogenic callus formation.
• Genotype:
• The genetic makeup of the plant species or cultivar significantly influences its ability to produce embryogenic callus.
• Explant Source:
• The type of explant used (e.g., leaf, stem, root) can affect the formation of embryogenic callus.
• Culture Medium:
• The composition of the culture medium, including nutrients and other additives, is essential for optimal embryogenic callus development.
• Capable of forming somatic embryos
• Usually yellowish-white or translucent
• Totipotent and can regenerate into whole plants

4. Non-embryogenic Callus

"Non-embryogenic callus" refers to callus tissue that lacks the capacity to produce somatic embryos. This distinguishes it from embryogenic callus, which is specifically characterized by its ability to develop into embryos. Here's a breakdown:

Key Characteristics:

• Lack of Somatic Embryogenesis:
• The defining feature of non-embryogenic callus is its inability to form somatic embryos, even when exposed to conditions that would induce embryogenesis in suitable callus tissue.
• Variable Morphology:
• Non-embryogenic callus can exhibit a range of textures, including friable (loose and crumbly) or compact (dense and firm).
• Different Cellular Structure:
• Compared to embryogenic callus, which often has cells with dense cytoplasm and prominent nuclei, non-embryogenic callus may have cells with larger vacuoles and a less organized structure.
• Different biochemical properties:
• There are differences in the protein and enzyme production in non-embryogenic callus, when compared to embryogenic callus.

Significance:

• Understanding Developmental Pathways:
• Studying non-embryogenic callus helps researchers understand the factors that control somatic embryogenesis. By comparing it with embryogenic callus, they can identify the genes and proteins involved in embryo development.
• Optimizing Tissue Culture Protocols:
• Understanding the conditions that lead to non-embryogenic callus formation is essential for optimizing tissue culture protocols and maximizing the production of embryogenic callus.
• Basic research:
• It is used in many basic research projects, to better understand plant cellular biology.
• Cannot form somatic embryos
• Used mainly for metabolite production or transformation

5. Organogenic Callus

Organogenic callus is a type of callus in plant tissue culture that has the capacity to develop into organized plant organs, such as shoots or roots. This is a key distinction from undifferentiated callus, which lacks this organizational potential. Here's a more detailed explanation:

Key Characteristics:

• Organ Formation:
• The defining characteristic is the ability to differentiate into specific plant organs. This process, known as organogenesis, can lead to the formation of shoots, roots, or even flowers.
• Organized Development:
• Unlike the random cell proliferation in undifferentiated callus, organogenic callus exhibits a degree of organized development, with cells forming distinct structures.
• Response to Hormones:
• The development of organs from organogenic callus is heavily influenced by the balance of plant growth regulators, particularly auxins and cytokinins, in the culture medium.
• Potential for Plant Regeneration:
• Organogenic callus is a valuable source for regenerating whole plants, particularly through the formation of shoots and roots.

Significance in Plant Tissue Culture:

• Plant Regeneration:
• It is a crucial tool for regenerating plants, especially in species that are difficult to propagate through other means.
• Micropropagation:
• Organogenic callus is used in micropropagation to produce large numbers of genetically identical plants.
• Genetic Transformation:
• It can serve as a target for genetic transformation, allowing for the introduction of desired genes into plants.
• Plant Breeding:
• It plays a role in plant breeding programs, facilitating the development of new plant varieties.
• Has potential to form organs like roots or shoots
• Often forms in response to specific PGR ratios

6. Pigmented Callus

Pigmented callus refers to callus tissue in plant tissue culture that exhibits coloration due to the production of various pigments. This coloration can vary significantly depending on the plant species, the specific pigments produced, and the culture conditions. Here's a more detailed explanation:

Causes of Pigmentation:

• Secondary Metabolites:
• Many plant species produce secondary metabolites, such as flavonoids, anthocyanins, carotenoids, and betalains, which can impart color to the callus.
• These compounds often serve protective functions in plants, such as UV protection or defense against pathogens.
• Chlorophyll:
• If the callus develops from tissues containing chloroplasts (e.g., leaves), it may exhibit a green coloration due to the presence of chlorophyll.
• Accumulation of Compounds:
• Sometimes the plant will accumulate different compounds within the callus, in response to the growth medium, or environmental conditions in the growth chamber.

Significance:

• Production of Valuable Compounds:
• Pigmented callus can be a valuable source for producing commercially important pigments or other secondary metabolites.
• For example, callus cultures of certain plants can be used to produce anthocyanins, which are used as natural food colorants and antioxidants.
• Visual Marker:
• Pigmentation can serve as a visual marker for specific developmental stages or the production of certain compounds within the callus.
• This is useful for research purposes.
• Research Tool:
• The study of pigmented callus can provide insights into the biosynthesis of plant pigments and other secondary metabolites.
• Genetic Studies:
• Pigmentation can also be used as a visual marker for genetic studies.

Examples:

• Callus cultures of certain species may exhibit red, purple, or blue coloration due to the accumulation of anthocyanins.
• Carotenoids can impart yellow, orange, or red coloration to callus tissue.
• Green callus is often observed when it originates from leaf tissue.

• May appear red, brown, purple due to pigment or stress
• Could indicate phenolic compound accumulation or senescence

Callus Characteristics to Observe

Type of Callus	Characteristics
Friable Callus	Soft, crumbly, loosely arranged; good for cell suspension culture
Compact Callus	Firm, dense, tightly packed; ideal for shoot/root regeneration
Embryogenic Callus	Appears granular; capable of forming somatic embryos and regenerating plants
Non-embryogenic Callus	Undifferentiated; lacks potential for embryo or organ development
Organogenic Callus	Shows early signs of shoot/root initiation; capable of forming organs
Pigmented Callus	Contains pigments (green, red, brown); may indicate specific metabolic activity
Mucilaginous Callus	Sticky, jelly-like due to mucilage secretion; common in species with high polysaccharide