Unit 3: Plant Biotechnology Practicals

Murashige & Skoog’s (MS) medium is the most widely used basal medium in plant tissue culture because it contains all the essential macro and micronutrients required for optimal growth.

Components of MS Medium

• Macronutrients: Include Nitrogen, Phosphorus, Potassium, Calcium, Magnesium, and Sulfur.
• Micronutrients: Essential trace elements like Iron, Manganese, Zinc, Boron, Copper, and Molybdenum.
• Carbon Source: Usually Sucrose (typically 3%), providing the energy necessary for cell metabolism.
• Vitamins and Organic Supplements: Such as Thiamine, Myo-inositol, and Glycine to support cellular health.
• Gelling Agent: Agar (0.8 - 1.0%) is added to solidify the medium for supporting the explant.

Preparation Steps

1. Dissolve stock solutions of macro and micronutrients in distilled water.
2. Add sucrose and mix thoroughly.
3. Adjust the pH to 5.8 using 0.1M HCl or NaOH.
4. Add agar and heat the mixture until it dissolves.
5. Sterilize the medium in an autoclave at 121°C (15 psi) for 15-20 minutes.

The success of tissue culture depends on using a healthy, sterilized starting material, called an explant.

Selection Criteria

• Choose young, actively growing tissues like shoot tips, axillary buds, or leaf discs as they have high regenerative potential.
• Ensure the parent plant is healthy and disease-free.

Surface Sterilization Procedure

1. Wash the explant under running tap water to remove surface dust.
2. Soak in a dilute detergent solution (e.g., Tween-20) for 5-10 minutes.
3. Rinse multiple times with distilled water.
4. Transfer to a Laminar Air Flow (LAF) chamber.
5. Surface sterilize using chemical agents like Mercuric Chloride (0.1%) or Sodium Hypochlorite (1-2%) for 3-5 minutes.
6. Rinse 3-4 times with sterile double-distilled water to remove all traces of chemicals.

Inoculation is the process of transferring the sterilized explant onto the culture medium under strictly aseptic conditions to prevent microbial contamination.

The Aseptic Environment

• Laminar Air Flow (LAF) Cabinet: Provides a sterile workspace by filtering air through HEPA filters.
• Personal Hygiene: Wash hands with 70% ethanol before starting work.
• Flame Sterilization: Dip tools like forceps and scalpels in 95% alcohol and flame them over a spirit lamp before each use.

Inoculation Procedure

1. Bring the sterilized explant and media containers into the LAF.
2. Open the culture vessel near the flame of a spirit lamp.
3. Carefully place the explant on the surface of the medium using sterilized forceps.
4. Close the vessel tightly and seal it with paraffin film.

Micropropagation is the rapid, large-scale clonal propagation of plants using tissue culture techniques.

Standard Stages

1. Stage 0: Selection and Preparation: Choosing the mother plant and preparing the explant.
2. Stage I: Establishment: Inoculation and initiation of culture in a sterile environment.
3. Stage II: Multiplication: Rapid division of shoots or embryos using specific hormone ratios (high cytokinin).
4. Stage III: Rooting: Transferring shoots to a rooting medium (high auxin) to develop roots.
5. Stage IV: Acclimatization (Hardening): Gradually moving the plantlets to greenhouse conditions to adapt them to the natural environment.

Plasmids are small, circular, extrachromosomal DNA molecules found in bacteria, used extensively as vectors in biotechnology.

Alkaline Lysis Method (General Steps)

1. Harvesting: Centrifuge the bacterial culture to collect the cell pellet.
2. Resuspension: Resuspend the pellet in a buffer containing EDTA (which destabilizes the cell wall).
3. Lysis: Add an alkaline solution (NaOH and SDS) to break the cells and denature DNA.
4. Neutralization: Add Potassium Acetate to precipitate genomic DNA and proteins, leaving smaller plasmid DNA in the solution.
5. Purification: Centrifuge to remove debris and precipitate the plasmid DNA using ethanol or isopropanol.

Protoplasts are plant cells that have had their cell walls removed, allowing for direct gene transfer or cell fusion.

Enzymatic Method

• Enzymes Used: Cellulase (to digest cellulose) and Pectinase (to digest the middle lamella/pectin).
• Osmoticum: Use of a high-concentration sugar solution (like 0.5M Mannitol) to prevent the "naked" cells from bursting due to osmotic pressure.
• Process: Incubate leaf tissues in the enzyme-osmoticum mixture until the cell walls are dissolved, then collect the spherical protoplasts by centrifugation.

7. Exam Focus: Practical Tips & Viva Prep

Frequently Asked Questions (Viva)

1. What is an explant? Any part of a plant (cell, tissue, or organ) used to initiate an in vitro culture.
2. Why is Agar used? As a solidifying agent because it is inert and not digested by plant enzymes.
3. What is Totipotency? The inherent ability of a single plant cell to regenerate into a whole plant.
4. Why is 70% Ethanol used for surface sterilization instead of 100%? 70% ethanol penetrates the microbial cell wall more effectively; 100% alcohol causes instant dehydration, creating a barrier that prevents the alcohol from entering and killing the microbe.

Common Pitfalls

• Contamination: The most common failure in PTC. Always ensure tools are properly flamed and the LAF is sterilized with UV light before use.
• pH Inaccuracy: If the pH is too high or too low, the agar will not solidify, and nutrients will not be available to the plant.