Unit 6: Protoplast Isolation - Subjective Questions

A protoplast is a plant cell from which the cell wall has been completely removed, leaving behind the naked cell bounded only by the plasma membrane, containing the cytoplasm, nucleus and organelles.

Importance in plant biotechnology:

• Somatic hybridization: Protoplasts from two different species can be fused to create somatic hybrids and cybrids, bypassing sexual incompatibility barriers.
• Genetic transformation: Naked protoplasts readily take up DNA, plasmids, and organelles, making them ideal for direct gene transfer (e.g., PEG-mediated, electroporation).
• Single-cell studies: They allow study of membrane transport, wall regeneration and cell division.
• Regeneration: A single protoplast can regenerate its wall and develop into a whole plant (totipotency).
• Mutant selection: Large populations of protoplasts can be screened for desirable mutants.

Mechanical Method of Protoplast Isolation:

This is the earliest technique used to isolate protoplasts, first demonstrated by Klercker (1892).

Procedure:

• Plant tissue is first placed in a suitable plasmolyticum (hypertonic solution such as 1 M sucrose).
• Plasmolysis causes the protoplasm to shrink away from the cell wall.
• The tissue is then cut or sliced with a sharp knife/razor.
• Deplasmolysis is induced, and protoplasts are released mechanically from cut ends of cells.

Advantages:

• No use of enzymes, so cells are not exposed to harmful enzymatic effects or osmotic stress from enzyme treatment.
• Useful for highly vacuolated cells (e.g., storage tissues like onion, radish, beet root).

Limitations:

• Very low yield of protoplasts.
• Laborious and tedious process.
• Restricted to certain tissues with large vacuolated cells.
• Cannot be used for tissues with meristematic or thin-walled cells.

Due to these limitations, the mechanical method has been largely replaced by the enzymatic method.

The enzymatic method, developed by Cocking (1960), is the most widely used technique for protoplast isolation because it gives a high yield of intact, viable protoplasts.

Principle: Cell wall components (cellulose, hemicellulose, pectin) are degraded by specific hydrolytic enzymes, releasing naked protoplasts.

Enzymes used:

• Cellulase – degrades cellulose.
• Hemicellulase – degrades hemicellulose.
• Pectinase (Macerozyme) – degrades the pectin of the middle lamella, separating cells.

Two approaches:

1. Two-step (sequential) method: Tissue is first treated with pectinase to separate cells (maceration), then cellulase to remove walls.
2. One-step (simultaneous) method: Tissue is treated with a mixture of pectinase + cellulase together. This is faster and more commonly used.

General procedure:

• Sterilize leaf tissue and peel off the lower epidermis.
• Incubate in an enzyme solution containing an osmoticum (e.g., mannitol/sorbitol 0.3–0.7 M) to prevent bursting.
• Incubate at 25–28°C for a few hours in the dark.
• Filter through a sieve to remove debris.
• Purify protoplasts by centrifugation (using sucrose gradient/floatation).
• Test viability and culture.

Advantages: High yield, large number of intact protoplasts, applicable to many tissue types.

Conclusion: The enzymatic method is preferred over the mechanical method because it produces a large number of viable protoplasts with minimal effort.

Role of Osmoticum:

When the cell wall is removed, the protoplast loses the mechanical support normally provided by the rigid wall. Without protection, the naked protoplast would burst (lyse) in a normal solution due to water entering by osmosis.

An osmoticum is an osmotic stabilizer added to the enzyme and culture medium to:

• Create a slightly hypertonic environment so that the protoplast remains plasmolysed and spherical.
• Prevent rupture of the fragile plasma membrane.
• Maintain osmotic balance during isolation, purification and culture.

Commonly used osmotica:

• Sugars/Sugar alcohols (metabolically inert): Mannitol, Sorbitol (most common, 0.3–0.7 M).
• Metabolically active sugars: Sucrose, Glucose.
• Salts: Sometimes CaCl, KCl are added for membrane stability.

Mannitol and sorbitol are preferred because they are not readily metabolized and provide stable osmotic pressure.

After enzymatic digestion, the crude protoplast suspension contains cell debris, undigested cells, vascular tissue, and broken protoplasts. Purification is essential to obtain a clean population of viable protoplasts.

Steps and Methods of Purification:

1. Filtration: The mixture is passed through a nylon/metal sieve (mesh size ~50–100 µm) to remove large debris and undigested tissue clumps.

2. Centrifugation (Sedimentation method):

   • The filtrate is centrifuged at low speed (~50–100 g).
   • Intact protoplasts settle at the bottom as a pellet, while debris remains in suspension.
   • The supernatant is discarded and pellet is resuspended.

3. Floatation method:

   • Protoplasts are suspended in a high-density sucrose solution.
   • On centrifugation, viable protoplasts float to the top while debris sediments.

4. Interphase (Density gradient) method:

   • A discontinuous gradient (e.g., sucrose over sorbitol/Ficoll) is prepared.
   • After centrifugation, intact protoplasts collect at the interface between the two layers as a distinct band.

Washing: Purified protoplasts are washed 2–3 times with culture medium to remove enzymes and osmoticum residues.

Testing viability determines whether isolated protoplasts are alive and capable of culture and regeneration.

Methods of Viability Testing:

1. Fluorescein Diacetate (FDA) Staining:

   • FDA is a non-fluorescent, non-polar compound that freely enters cells.
   • Inside living cells, esterases cleave it to release fluorescein, which accumulates and fluoresces green under UV/fluorescence microscope.
   • Viable protoplasts fluoresce green; dead ones do not.

2. Phenosafranine Staining:

   • This dye is taken up only by dead protoplasts, staining them red.
   • Viable protoplasts remain unstained.
   • Simple and rapid exclusion test.

3. Evan's Blue / Trypan Blue Exclusion:

   • Living cells with intact membranes exclude the dye and remain colourless.
   • Dead cells take up the dye and appear blue.

4. Other indicators: Observation of cytoplasmic streaming, oxygen uptake / respiration, and the ability to undergo cell wall regeneration and division also confirm viability.

Percentage viability is calculated as:

The plant cell wall is a complex structure made of cellulose, hemicellulose, and pectin. Different enzymes target these specific components:

1. Cellulase:

• Degrades cellulose, the main structural polysaccharide (β-1,4-glucan chains) of the cell wall.
• Commercial example: Cellulase Onozuka R-10.

2. Hemicellulase:

• Degrades hemicellulose, the matrix polysaccharide binding cellulose microfibrils.
• Example: Rhozyme HP-150.

3. Pectinase (Macerozyme / Pectolyase):

• Degrades pectin of the middle lamella, the cementing layer between adjacent cells.
• This causes maceration (cell separation) into individual cells.
• Example: Macerozyme R-10, Pectolyase Y-23.

Additional notes:

• Enzymes are usually of fungal origin (from Trichoderma, Aspergillus, etc.).
• They are used at pH 4.7–6.0 and require an osmoticum in the solution.
• A combination (cellulase + pectinase) is used in the one-step method for complete wall removal.

Several factors influence the yield, viability and quality of isolated protoplasts:

1. Source Tissue:

• Young, actively growing tissues (e.g., mesophyll of young leaves) give better yields.
• Physiological state and age of the plant matter greatly.

2. Enzyme Concentration and Combination:

• Optimal concentration of cellulase and pectinase must be used.
• Excess enzyme or prolonged exposure damages protoplasts.

3. Incubation Time and Temperature:

• Usually 25–30°C for a few hours; over-incubation reduces viability.

4. pH:

• Enzyme activity is optimal around pH 4.7–6.0.

5. Osmoticum:

• Correct concentration (0.3–0.7 M mannitol/sorbitol) prevents bursting.

6. Light:

• Incubation is generally done in dark or dim light to reduce damage.

7. Plant genotype and growth conditions:

• Greenhouse-grown, healthy plants give better protoplasts than field-grown plants.

8. Presence of stabilizing salts:

• CaCl and other ions improve membrane stability.

Optimizing all these factors together maximizes both quantity and quality of protoplasts.

Protoplast Fusion (Somatic Hybridization):

It is the process of fusing protoplasts of two genetically different cells to produce a hybrid cell (heterokaryon), which can regenerate into a somatic hybrid plant.

Methods of Fusion:

• Spontaneous fusion: Occurs naturally during isolation via plasmodesmata.
• Induced fusion:
  • Chemical (PEG-mediated): Polyethylene glycol induces adhesion and fusion of membranes.
  • Electrofusion: An electric field aligns protoplasts (pearl-chain) and a pulse fuses them.
  • Other chemicals: NaNO, high pH/high Ca solution.

Products of Fusion:

• Hybrid: Fusion of nuclei and cytoplasm from both parents.
• Cybrid: Cytoplasm from both parents but nucleus from only one.

Significance:

• Overcomes sexual incompatibility barriers between species.
• Enables transfer of cytoplasmic traits (e.g., cytoplasmic male sterility, disease resistance).
• Produces novel hybrids not possible by conventional breeding.
• Useful in crop improvement and germplasm development.

Once isolated and placed in a suitable culture medium, viable protoplasts begin to regenerate a new cell wall and eventually divide — demonstrating their totipotency.

Stages:

1. Cell Wall Regeneration:

• Within a few hours to days, protoplasts start depositing cellulose microfibrils on the plasma membrane surface.
• Wall regeneration can be detected using Calcofluor white staining (fluoresces on cellulose).
• The protoplast gradually changes from spherical to oval shape as the rigid wall forms.
• Once the wall is regenerated, the structure is called a cell (no longer a protoplast).

2. Cell Division:

• After wall formation, the cell undergoes its first mitotic division (usually within 2–7 days).
• Repeated divisions form a cell colony / microcallus.

3. Callus and Regeneration:

• Continued division produces a callus.
• Under appropriate hormone balance (auxin:cytokinin), the callus differentiates into shoots and roots, regenerating into a whole plant (organogenesis or embryogenesis).

This sequence — protoplast → wall regeneration → division → callus → plantlet — confirms the viability and totipotency of the isolated protoplasts.

Isolated and purified protoplasts are cultured in nutrient media to encourage wall regeneration, division and regeneration. The main culture techniques are:

1. Liquid (Suspension) Culture:

• Protoplasts are suspended in a thin layer of liquid medium in petri dishes.
• Allows easy dilution, medium change, and osmoticum reduction.
• Good for aeration but risk of clumping.

2. Agar (Solid) Culture / Bead Type Culture:

• Protoplasts are mixed with molten agar/agarose medium and allowed to solidify.
• Each protoplast is fixed in position, preventing clumping and allowing tracking of single-cell development.
• Agarose bead culture: protoplasts embedded in agarose beads floated in liquid medium.

3. Feeder Layer Technique:

• A layer of X-ray irradiated (non-dividing) nurse cells provides growth factors to low-density protoplasts.
• Useful for culturing protoplasts at very low densities.

4. Co-culture / Nurse Culture:

• Actively growing cells are cultured along with protoplasts to supply conditioning factors.

5. Micro-drop / Micro-chamber Culture:

• Individual protoplasts cultured in tiny droplets for single-cell cloning.

Medium: Commonly modified MS or KM (Kao & Michayluk) medium with osmoticum and appropriate hormones is used.

Since protoplasts are naked cells highly susceptible to contamination and damage, strict aseptic and careful handling is essential.

Sterilization / Aseptic Precautions:

• Surface sterilization of plant material using 70% ethanol followed by sodium hypochlorite (NaOCl) and rinsing with sterile distilled water.
• All glassware, media and enzyme solutions must be sterilized (enzyme solutions filter-sterilized through 0.22 µm membrane as they are heat-labile).
• Work carried out inside a laminar air flow cabinet.

Handling Precautions:

• Use gentle centrifugation (low speed) to avoid rupturing protoplasts.
• Maintain proper osmoticum concentration to prevent bursting.
• Avoid mechanical stress (vigorous pipetting/shaking).
• Control enzyme concentration and incubation time to prevent over-digestion.
• Maintain optimal pH and temperature.
• Work quickly to reduce exposure of protoplasts to enzymes.

Reason: Any contamination or mechanical/osmotic injury drastically reduces yield, viability, and success of subsequent culture.

Both are enzymatic approaches, differing in how the enzymes are applied.

Two-step method is useful when isolated cells are also needed, or for delicate tissues.

One-step method is preferred for routine work due to its simplicity and speed, as both maceration and wall digestion occur simultaneously.

Protoplast technology has wide-ranging applications:

1. Somatic Hybridization:

• Fusion of protoplasts to create interspecific/intergeneric hybrids overcoming sexual incompatibility (e.g., Pomato = potato + tomato).

2. Cybrid Production:

• Transfer of cytoplasmic traits like cytoplasmic male sterility (CMS) and herbicide/disease resistance.

3. Genetic Transformation:

• Naked protoplasts easily take up foreign DNA, plasmids, viruses, and organelles (via PEG, electroporation, microinjection) for producing transgenic plants.

4. Organelle and DNA Uptake:

• Study and transfer of chloroplasts, mitochondria and nuclei between cells.

5. Mutant Selection:

• Millions of protoplasts can be screened to select useful mutants (disease/stress resistant).

6. Study of Basic Cell Biology:

• Investigation of membrane properties, wall regeneration, cell division, and virus infection mechanisms.

7. Production of Virus-free Plants:

• Protoplast culture can help obtain disease-free clones.

8. Germplasm Conservation:

• Protoplasts can be cryopreserved for storage.

Thus, protoplasts are a powerful tool for crop improvement and plant genetic engineering.

1. PEG (Polyethylene Glycol) Mediated Fusion (Chemical Method):

• PEG is a chemical fusogen that induces protoplasts to adhere and fuse.
• Mechanism: PEG causes membrane disturbance and tight adhesion between protoplasts; on washing/dilution (often with high Ca, high pH), the membranes fuse.
• Advantages: Simple, inexpensive, fuses large populations, reproducible.
• Disadvantages: PEG can be toxic to some protoplasts; fusion is random (non-specific).

2. Electrofusion (Physical Method):

• Protoplasts are placed in a fusion chamber between electrodes.
• A weak alternating current (AC) causes protoplasts to align into a pearl-chain (dielectrophoresis).
• A brief high-voltage direct current (DC) pulse causes reversible membrane breakdown (electroporation) at contact points, leading to fusion.
• Advantages: Precise, controllable, high fusion frequency, non-toxic, no chemicals.
• Disadvantages: Requires specialized equipment, higher cost.

Comparison: PEG is cheaper and simpler but chemically harsh; electrofusion is gentler and more controlled but requires expensive apparatus.

The enzyme solution used for protoplast isolation is carefully formulated to digest the cell wall while protecting the protoplast.

Components and their roles:

1. Cell wall degrading enzymes:

• Cellulase – digests cellulose.
• Pectinase / Macerozyme – digests pectin of the middle lamella.
• Hemicellulase – digests hemicellulose.

2. Osmoticum (osmotic stabilizer):

• Mannitol / Sorbitol (0.3–0.7 M) – prevents protoplast bursting by maintaining osmotic balance.

3. Buffer:

• MES buffer to maintain optimal pH (~5.5–5.8) for enzyme activity.

4. Salts / Membrane stabilizers:

• CaCl and other salts stabilize the plasma membrane and improve protoplast integrity.

5. Sometimes:

• Bovine serum albumin (BSA) or potassium dextran sulphate to protect protoplasts.
• Antibiotics to reduce contamination.

Preparation notes:

• The solution is filter-sterilized (enzymes are heat-labile).
• pH is adjusted to enzyme optimum before use.

Proper balance of these components ensures efficient wall digestion with high protoplast viability.

Although protoplast technology is powerful, several problems and limitations exist:

1. Genotype dependency:

• Success of isolation and regeneration varies greatly between species; cereals and legumes are often recalcitrant.

2. Low viability / damage:

• Enzymatic treatment, osmotic stress and mechanical handling can damage protoplasts, reducing viability.

3. Difficulty in wall regeneration and division:

• Some protoplasts fail to regenerate walls or divide.

4. Somaclonal variation:

• Regenerated plants may show genetic/chromosomal abnormalities and undesirable variation.

5. Contamination risk:

• Naked protoplasts are highly susceptible to microbial contamination.

6. Albinism / abnormal plants:

• Regenerated plants (especially in cereals) may be albino or infertile.

7. Random fusion:

• In somatic hybridization, fusion is non-specific, requiring selection of true hybrids.

8. Time-consuming and technically demanding:

• Requires optimization of many parameters and skilled handling.

Despite these limitations, continuous refinement of media and techniques has improved success in many crop species.

The development of protoplast isolation techniques progressed through key contributions:

1. Klercker (1892):

• First isolated protoplasts using the mechanical method by plasmolysing and cutting plant tissue.
• Yield was very low and the method laborious.

2. E.C. Cocking (1960):

• Father of protoplast research.
• First successfully isolated protoplasts using the enzymatic method (cellulase from fungal source) from tomato root tips.
• This revolutionized the field by giving large numbers of viable protoplasts.

3. Takebe et al. (1968, 1971):

• Used commercially available enzymes and achieved isolation from tobacco mesophyll.
• Demonstrated regeneration of whole tobacco plants from protoplasts, proving totipotency.

4. Carlson et al. (1972):

• Produced the first somatic hybrid by fusing protoplasts of two Nicotiana species.

5. Later developments:

• Refinement of enzyme mixtures, culture media (Kao & Michayluk medium), and fusion techniques (PEG, electrofusion) expanded applications.

These milestones transformed protoplast isolation from a difficult laboratory curiosity into a standard biotechnological tool.

Protocol for Enzymatic Isolation of Protoplasts from Tobacco Leaf Mesophyll:

Step 1 – Selection of material:

• Take healthy, young, fully expanded leaves from a well-grown tobacco plant.

Step 2 – Surface sterilization:

• Wash leaves, dip in 70% ethanol (30 sec), then in sodium hypochlorite solution (a few minutes), and rinse 3–4 times with sterile distilled water.

Step 3 – Peeling / slicing:

• Peel off the lower epidermis to expose mesophyll cells (or cut leaf into thin strips).

Step 4 – Pre-plasmolysis:

• Float leaf pieces in a plasmolyticum (mannitol solution) for about 1 hour to induce plasmolysis (reduces damage).

Step 5 – Enzyme treatment:

• Transfer pieces into enzyme solution containing cellulase + macerozyme, osmoticum (mannitol 0.5–0.7 M), MES buffer (pH ~5.7) and CaCl.
• Incubate at 25–28°C in the dark for 3–6 hours with gentle shaking.

Step 6 – Filtration:

• Filter the digest through a nylon sieve (~50–100 µm) to remove debris.

Step 7 – Purification:

• Centrifuge at low speed (~50–100 g). Purify by floatation/density gradient to obtain a clean band of protoplasts.

Step 8 – Washing:

• Wash 2–3 times with washing/culture medium to remove enzymes and osmoticum.

Step 9 – Viability test:

• Test viability using FDA staining and count using a haemocytometer.

Step 10 – Culture:

• Suspend purified viable protoplasts in suitable medium and culture for wall regeneration and division.

Result: A pure suspension of viable tobacco mesophyll protoplasts ready for culture or fusion.