Protoplast isolation, culture, application

To understand the protoplast isolation, lets Imagine being able to take two completely different plants—one resistant to drought, the other producing high-yield crops—and fusing them into a single super-plant.

What if you could manipulate individual cells and create hybrids that nature never could? Welcome to the fascinating world of protoplast isolation, where we strip cells down to their essence and unlock limitless possibilities for hybridization and crop improvement!

So, let’s dive into it and first understand,

What is protoplast?

“Protoplasts are plant cells where the cell wall has been enzymatically removed, leaving only the plasma membrane intact.”

• These are derived from plant tissues through the use of enzymes such as cellulase and pectinase, which digest the rigid cell walls.
• Once the cell wall is removed, protoplasts are essentially naked cells with their internal organelles, including the nucleus, chloroplasts, and vacuoles, surrounded by a plasma membrane.
• Significance: Serve as essential units in cellular biology, housing genetic information crucial for various cellular processes (reproduction, differentiation, regeneration).
• Lack of Cell Wall: Enhances the uptake of external genetic material, making them ideal for genetic transformation studies.

Why protoplast are preferred for genetic manipulation?

The –nt of cell wall makes the protoplast suitable for variety of experimental manipulation which are not possible with intact cells

Ideal material for injecting foreign material like DNA, Mitochondria, chloroplast for genetic manipulation.

Now we know the protoplast, but

what are the methods of protoplast isolation?

There are two methods to isolate protoplast from plant tissue-

1. Mechanical Methods
2. Enzymatic Methods

Mechnical method of protoplast isolation

In the mechanical method, protoplasts are isolated by using physical disruption of plant tissues to release them.

This technique typically employs mechanical forces, such as grinding or chopping, to break down the cell walls of plant cells, allowing the protoplasts to be extracted.

Steps of mechanical method of protoplast isolation –

• Explant (leaf, roots, patels etc.) were scratched to remove epidermis
• Surface sterilization
• Plasmolysis by 10M sucrose
• Then cut the cell wall using knife or grinding.
• Release of protoplast
• Collection of protoplasts

Limitation:

• It is labor-intensive
• Yields low number of intact protoplasts.
• High risk of damaging protoplasts.

Enzymatic method of isolation of protoplast

If we look into the tissue structure of plant, we will find the adjacent cells are attached together by pectin and the plant individual cell is surround by cell wall.

So In the enzymatic method of isolation, this cell-wall-degrading or pectin degrading enzymes are used to release protoplasts from plant tissues in a more controlled and efficient manner.

There are two ways to use these enzymes for the isolation of protoplast

1. One step enzymatic method : in this method the pactinase and cellulase enzyme were used in appropriate mixture.
2. Two step enzymatic method :  It is carried out in two steps, in 1^st plant tissue is given pactinase treatment to separate the individual cells in tissue explants. Then in 2^nd steps separated cells were treated with cellulase to remove the cell-wall.

Steps of enzymatic method of isolation of protoplast –

• Sterilization of the Explant (root, leaf etc.).
• Remove epidermis
• Plant tissue is immersed in a solution containing cellulase (breaks down cellulose) and pectinase (breaks down pectin) to digest the cell walls either together or separately.
• The enzyme solution is incubated for several hours, allowing the cell wall/pectin to be completely dissolved.
• The tissue is then gently agitated to release the protoplasts into the solution.
• Protoplasts are collected and purified through filtration/centrifugation.

Viability testing after isolation of protoplast

There are different techniques available to check viability of isolated protoplast/ or cells. some of the techniques are as –

• Fluoreciein diacetate staining – inside the cells esterases in viable cells convert it into fluorescein give fluorescence in living cells but not dead.
• Tetrazolium chloride staining – detect metabolic activity. Stain get reduced by dehydrogenase enzymes in living cells to form a red-colored formazan compound. Dead cells remain colorless.
• Evan’s Blue – It is a dye that penetrates cells with damaged membranes (non-viable cells). Life cells do not stain.
• Phenosaffranine staining– Stain dead cells give red color but not living cells.
• Calcofluor white staining – binds to beta-linked glucosides in newly formed cell wall and gives fluorescence around plasma membrane.

Protoplast culture

To culture protoplast there are many ways. It can be cultured in Agar medium or in the liquid medium plates.

• But we may need to change composition of culture medium as need. In general, when we are making culture medium it should have no ammonium as it is detrimental to the protoplast.
• L-glutamine / Casin hydrolysate can be used to meat satisfactory growth if N source/ amino acid need.
• There should be low amount of Fe and Zn metal source for good growth.
• Ca++ should be used in double amount compare to normal medium. it is good for membrane stability.
• Mannitol / sorbitol use to maintain osmolarity.
• Glucose as carbon source alone or with sucrose but not sucrose alone.
• Groth regulator Auxin and cytokinin.
• temprature range – 20-28 degrees C.
• pH range = 5.5-5.9

Techniques of protoplast culture

To culture protoplast there are many different methods based on medium (with or without agar) and their placement in it-

• Agar culture technique: in this technique the protoplast suspension is mixed with agar containg medium molten at 45 degrees Celsius. Plates are allowed to solidify and sealed with parafilm in incubated.
• liquid droplet method: liquid medium is uses in this method. Protoplast suspension is pipeted out (approx- 100-200 ul) into the Patri plates as drop and then incubated.
• Hanging drop method: This similar to liquid droplet method but in this method drops are placed into the lid of Petri dish. When the lid containing drop of culture is applied to the plate of petri plate it appears hanging.
• Feeder layer method- this method involves cultureing the protoplast on layer of non-dividing nurse cells (called feeder layer).
• Co-culture method – two different types of protoplasts is used for the culture. one is fast and other is slow.

Factors affecting of protoplast isolation

Several factors influence the isolation of protoplast-

• Restricted to highly vascularized tissues in case of Mechanical method.
• Choice of enzyme: Depends on the cell wall composition of the plant tissue.
• Enzyme concentration: Affects the efficiency and speed of cell wall digestion.
• Osmotic pressure: Must be controlled to maintain protoplast integrity during the isolation process.
• Enzymatic digestion temperature: Optimal temperature is critical for enzyme activity and protoplast viability.
• Duration of digestion: Prolonged exposure to enzymes can damage protoplasts, while insufficient digestion reduces yield.

Application of protoplast isolation and culture

• Plant Regeneration:
  • Protoplasts can be regenerated into whole plants through tissue culture techniques. This is important for clonal propagation, allowing large-scale multiplication of plants with desirable traits.
• Study of Cell Biology:
  • Protoplasts serve as a model system for studying cellular processes like membrane transport, cell wall formation, and gene expression, helping in fundamental plant biology research.
• In Vitro Breeding:
  • Protoplast culture is used for in vitro selection of plants with resistance to various abiotic stresses (e.g., salt tolerance, drought resistance) and for mutation breeding programs.
• Somatic Hybridization:
  • Protoplast fusion enables the creation of somatic hybrids by fusing protoplasts from different plant species or varieties, even those that are sexually incompatible. This is valuable in plant breeding to develop new hybrids with desired traits.
• Genetic Modification:
  • Protoplasts are ideal for introducing foreign genes into plant cells via techniques like direct DNA uptake or Agrobacterium-mediated transformation. This allows the creation of genetically modified plants (GMOs) with enhanced traits, such as pest resistance, Disease Resistance or improved nutritional content.
• Cybridization:
  • This technique involves fusing protoplasts from two different species to create cybrids (cytoplasmic hybrids) that combine nuclear material from one parent and cytoplasmic (mitochondria and chloroplasts) material from another, aiding in the transfer of cytoplasmic male sterility for hybrid seed production.
•  Secondary Metabolite Production:
  • Isolated protoplasts can be used in culture to study and enhance the production of secondary metabolites, such as alkaloids and flavonoids, which have pharmaceutical and industrial value.

FAQs

Reference

Chen K, Chen J, Pi X, Huang LJ, Li N. Isolation, Purification, and Application of Protoplasts and Transient Expression Systems in Plants. Int J Mol Sci. 2023 Nov 29;24(23):16892. doi: 10.3390/ijms242316892. PMID: 38069215; PMCID: PMC10706244.

Chawla, H. S. (2002). Introduction to plant biotechnology (3rd ed.). Oxford & IHB Publishing Co. Pvt. Ltd. New Delhi; ISBN 1978-81-204-1732-8

Ahmed, M. A., Miao, M., Pratsinakis, E. D., Zhang, H., Wang, W., Yuan, Y., … & Wu, B. (2021). Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp. Agriculture, 11(8), 699.