14 Jun 2024

Understanding Plant Growth Regulators: Types and Uses in Tissue Culture

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplify intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Introduction

Ever wondered how plants develop their stems, roots, and leaves? 

Tiny chemical messengers called plant growth regulators (PGRs) play a crucial role in this process. 

Like us humans with hormones, plants use PGRs to grow and develop. 

These molecules influence activities like seed germination, organ development, flowering, and even aging.

Do you know, how scientists define these molecules,?

Here is how one study explains PGRs: “Plant growth regulators are naturally biosynthesized chemicals in plants that influence the physiological process.” 

Too jargony, right?

But, simply, it’s saying the same thing I explained to you before. 

Plant growth regulators (PGRs) are natural or man-made chemical messengers that influence how plants grow and develop.

There are two main types of PGRs:

• Natural PGRs (phytohormones): Produced by plants themselves and regulate various growth processes.
• Synthetic PGRs: Man-made versions or entirely new compounds that mimic natural PGRs.

Both can trigger important changes in plants, affecting everything from cell division and stem elongation to flowering and fruit development. 

In this article, we'll explore the different classes of PGRs, how they're made, and their specific functions within plants. We'll also explore how some of these hormones are used in plant tissue culture applications.

Classes of Plant Growth Regulators 

Auxin, cytokinin, gibberellins, ethylene, and abscisic acid are the well-known major classes of plant growth regulators. However, there's more to the story!

Plants also utilize a fascinating array of lesser-known plant hormones that significantly contribute to their development.

These include:

• Brassinosteroids: These hormones promote cell division, stem elongation, and stress tolerance.
• Strigolactones: These versatile players influence root development, shoot branching, and communication with beneficial soil microbes.
• Jasmonates: Often associated with plant defense, jasmonates also regulate growth processes and responses to environmental cues.
• Salicylic acid: It acts as a natural defense system, helping plants fight off disease-causing pathogens.

Understanding these diverse plant growth regulators is crucial for unlocking a deeper understanding of plant growth and development.

Hormones Used in Plant Tissue Culture

We know nine classes of plant hormones, however, mostly three classes of these hormones are frequently used in tissue culture processes. It includes: auxin, cytokinin, and gibberellins.

Auxin

Did you know the secret to a plant's bending towards light or a flower's vibrant bloom lies within a tiny molecule?

 It's auxin—a well-known plant hormone involved in the growth and development of plants.

Discovery

As early as the late 1800s, Charles Darwin and his son observed a curious phenomenon—grass coleoptiles bending towards light.  Their experiments hinted at a mysterious chemical signal traveling from the tip, influencing stem growth. 

Fast forward to the 1920s, Frits Went, a botanist, isolated this very substance from oat shoot tips, confirming its growth-stimulating power. Consequently, this "growth factor" was aptly named auxin, derived from the Greek word "auxano" meaning "to grow."

Biosynthesis

Plants primarily manufacture auxin in their apical buds (growing tips) of stems and roots. Here's a glimpse into the two major pathways involved:

• Tryptophan-dependent pathway: The most common route, it utilizes the amino acid tryptophan as its starting point. Then, through a series of enzymatic steps, tryptophan is converted into IAA.
• Tryptophan-independent pathway: Less well-understood, this pathway utilizes other precursors besides tryptophan to generate auxin. However, the exact details are still being unraveled.

Structure

IAA is a simple molecule with a ring-like structure, but its effects on plant development are far-reaching.

Function

• Cell Elongation: It promotes elongation of plant cells, influencing stem growth and root formation.
• Apical Dominance: Auxin ensures a plant's dominant upward growth by inhibiting the growth of lateral buds. Imagine a plant without auxin - it would be a bushy mess!
• Phototropism: Auxin is the secret sauce behind a plant's bending towards light. It accumulates on the shaded side of a stem, stimulating faster cell elongation there, causing the plant to bend towards the light source.
• Fruit Development: Auxin plays a role in fruit development, influencing cell division and fruit set.

Application in Tissue Culture

Auxin's ability to promote root growth makes it a cornerstone of plant tissue culture. Scientists utilize synthetic auxins to encourage root formation in stem cuttings and plantlets grown in controlled environments.

Here's how different types of auxins are utilized:

• Indole-3-acetic acid (IAA): It's a primary auxin. It's widely used in plant tissue culture to stimulate cell division and elongation, promoting the formation of roots in explants. Its application enhances callus formation, organogenesis, and the overall growth and development of cultured tissues.
• Indole-3-butyric acid (IBA): This synthetic auxin excels at stimulating root initiation in cuttings and plantlets. Its effectiveness lies in its ability to readily enter plant cells and trigger root formation.
• 1-Naphthaleneacetic acid (NAA): Another synthetic auxin, NAA is often used in combination with cytokinins (another plant hormone group) to promote shoot proliferation and root development in callus cultures (undifferentiated plant cell masses).
• 2,4-Dichlorophenoxyacetic acid (2,4-D): This synthetic auxin has a wider range of applications, influencing cell division, root initiation, and embryogenesis (formation of embryos) in tissue culture. However, its use is often restricted due to environmental concerns.

By strategically applying these auxin types, scientists can manipulate plant growth and development in a controlled tissue culture environment, paving the way for rapid plant propagation and genetic improvement.

Cytokinin

Cytokinins, a class of plant hormones, are the masters of cell division and development, playing a vital role in plant growth from seed to bloom. 

Discovery

Early Observations (1920-1940): Haberlandt and van Overbeek discovered diffusible substances in plant tissues and coconut milk that stimulated cell division, hinting at the existence of cytokinins.

Breakthrough Isolation (1955): Skoog and colleagues isolated and crystallized a "cell division factor" from herring sperm DNA, naming it kinetin. This discovery sparked the search for natural cytokinins in plants.

Unveiling the Natural Powerhouse (1963): Letham isolated zeatin, the first natural cytokinin, from corn kernels. This, along with subsequent discoveries, established cytokinins as a unique hormone class.

Biosynthesis

Cytokinins are primarily produced in actively growing plant parts like root tips, shoot tips, young leaves, and seeds.

Here's a glimpse into the major pathways involved:

• Methylerythritol Phosphate (MEP) Pathway: This pathway occurs in the plastids and is responsible for the production of the isoprenoid precursor, isopentenyl diphosphate (IPP).
• tRNA-Isopentenyltransferase Pathway: A parallel pathway involves the modification of certain tRNAs by tRNA-isopentenyltransferases (tRNA-IPTs), where cytokinins are produced as side products:

• tRNA-IPT enzymes add isopentenyl groups to adenosine residues in tRNAs.
• Degradation of these modified tRNAs releases free cytokinins.

Structure

These hormones vary in structure, but often share an adenine (purine) base with a side chain.

Function

Cell Division and Differentiation: They trigger cell division and influence cell differentiation, shaping plant structures.

Organogenesis: Cytokinins promote the formation of new organs like shoots and roots.

Delaying Leaf Senescence: They keep leaves youthful by delaying the aging process.

De-etiolation Response: Cytokinins help seedlings overcome darkness by promoting chlorophyll production and greening.

Application in Tissue Culture

Here’s how different cystokinins are utilized in tissue culture applications:

• Benzyladenine (BA): This synthetic cytokinin excels at stimulating cell division and shoot bud formation. Its water solubility and ease of use make it a popular choice for diverse tissue culture applications.
• Kinetin: The "cell division factor" discovered from herring sperm DNA, kinetin is a potent inducer of cell division and shoot proliferation. It's often used in combination with auxins for efficient shoot regeneration.
• Zeatin: The first natural cytokinin discovered, zeatin closely mimics the effects of naturally occurring cytokinins in plants. It's often used for shoot proliferation and bud break, particularly for plant species sensitive to synthetic cytokinins.

Gibberellins

A gibberellin hormone (GA) is a plant hormone that plays a vital role in various growth and developmental processes throughout a plant's life cycle.

Discovery

The story of gibberellins is quite interesting. They were first identified around 75 years ago from a fungus called Gibberella fujikuroi, which causes a strange rice disease called "bakanae" (foolish seedling disease) by overproducing GAs. Scientists later discovered that controlled application of GAs could actually benefit plants!

Biosynthesis

Plants manufacture gibberellins primarily in their young tissues like growing buds and developing seeds. Here's a glimpse into the two major pathways involved in their syntesis:

• Mevalonate (MVA) Pathway: This pathway, commonly found in plants, utilizes the precursor molecule acetyl-CoA to build the basic building blocks for GAs. Through a series of enzymatic steps, these precursors are then transformed into the core GA structure.
• Methylerythritol phosphate (MEP) Pathway: This alternative pathway, also present in plants, starts with a different precursor molecule and utilizes a distinct set of enzymes to arrive at the GA core structure.

Both pathways converge at a specific point, leading to the final creation of various gibberellin types through further modifications.

Structure

Gibberellins are a diverse group of molecules, but they often share a core structure based on a tetracyclic diterpene backbone. This intricate structure allows GAs to interact with specific plant proteins, triggering a cascade of events that promote growth.

Function

Gibberellins are essential for promoting numerous growth processes in plants, including:

• Seed Germination: They break seed dormancy, signaling the embryo to initiate germination and sprout a new plant.
• Stem Elongation: GAs stimulate cell elongation, leading to taller and stronger stems.
• Flowering: They influence the timing and development of flowers in some plants.
• Leaf Expansion: GAs promote leaf growth, maximizing a plant's ability to photosynthesize and produce energy.
• Other Functions: GAs can also influence fruit development, stress resistance, and enzyme activity in plants.

Application in Tissue Culture

While research on GA applications in tissue culture is ongoing, these versatile hormones show promise in specific areas:

• Overcoming Seed Dormancy: Certain GAs, like GA3 (gibberellic acid 3), can mimic the natural signal to break seed dormancy in some plant embryos, facilitating germination within a controlled tissue culture environment. This allows scientists to bypass natural dormancy periods and accelerate plant propagation.
• Stem Elongation: In specific protocols, applying low concentrations of GAs can promote controlled stem elongation in certain plantlets grown in tissue culture. This can be beneficial for developing stronger and more robust plantlets before transplantation.

How To Prepare Stock Solutions of Plant Growth Regulators?

Follow the given steps to prepare the stock solution of your desired plant hormones. 

Steps:

1. Measure the PGR: Using your balance, carefully weigh out the required amount of PGR powder to achieve a 1 mg/mL stock solution. For example, to prepare a 100 mL stock solution, you'd need 100 mg of PGR powder.

2. Dissolve the PGR: Add 5-7 mL of a suitable solvent to the volumetric flask containing the weighed PGR powder. Common solvents for PGRs include ethanol, methanol, or a very small amount of 1 N NaOH or KOH (depending on the specific PGR).

Important Note: Always consult the specific PGR's safety data sheet (SDS) to determine the appropriate solvent and safety precautions for handling.

3. Swirl: Swirl the flask gently to aid in dissolving the PGR. 

4. Bring to Volume: Once the PGR is completely dissolved, slowly add distilled or deionized water to the flask while swirling to ensure even mixing. Fill the flask to the etched line marking the desired volume (usually 100 mL).

5. Optional Stirring: For better homogeneity, you can use a stir bar or stir the solution gently with a clean stirring rod.

6. Labeling: Affix a label to the flask containing the following information:

   • Name of the PGR
   • Stock solution concentration (e.g., 1 mg/mL)
   • Date of preparation
   • Your initials

7. Storage: Store the prepared stock solution in a cool, dark place (typically a refrigerator at 4°C) unless otherwise specified for the specific PGR.

Remember:

• This is a general guide for preparing a 1 mg/mL stock solution. The required concentration and solvent might vary depending on the specific PGR you're using.
• Always refer to the manufacturer's instructions or relevant scientific literature for specific details.

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