What is Protoplast?
Protoplast is the plant cell without a cell wall. This protoplast possesses other cellular components and a plasma membrane. Hence, this protoplast is a functional plant cell without a barrier i.e. cell wall.
Steps involved in protoplast culture
- Sources of protoplast
- Isolation of protoplast
- Purification of protoplast
- Viability check of protoplasts
- Culture of protoplasts
1. Source of protoplast: Tissues and organs like leaves, roots, shoot apices, fruits, embryos and microspores are widely used as sources for protoplast. Callus and suspension cultures are also good sources for protoplast isolation.
2. Isolation of protoplast: It can be done by two methods
a) Mechanical method: In this method, the cells of the epidermis are subjected to plasmolysis, by which protoplast causes to shrink away from the cell wall. Then, the protoplast is released by tissue dissection. The main disadvantage of this method that, the yield and viability of protoplast is very low. This process is tedious and laborious.
b) Enzymatic method: This method has an advantage over the mechanical method, which has a high yield of protoplasts with viable cells. Hence, it is a widely used method. Enzymes like cellulases, hemicellulases, pectinases, are used to degrade cellulose, hemicellulose, and pectin in the plant cell wall.
One of the two methods is used to isolate protoplast by enzymatic method.
- Two-step method: in this method the tissue is treated with pectinase to degrade middle lamella, to separate cells from tissue. These free cells are treated with cellulose to remove the cell wall, which results in the release of protoplast.
- One-step method: in this method the tissue is treated simultaneously with cellulose and pectinase. Hence it is the most preferred method.
3. Purification of protoplast: It is done by filtration to remove cell clumps and undigested tissues, followed by centrifugation and washing of protoplast.
4. Viability test of protoplast: The following are the methods used to test the viability of protoplast
a) Testing photosynthetic activity of protoplast
b) Measurements of cell wall formation can be observed using Calcofluor white (CFW) stain, which bind to the newly formed cell wall and emits fluorescence.
c) Measurement of oxygen uptake by protoplast by using oxygen electrode
d) Phenosafranine stain: dead protoplast uptake this stain and turns red, whereas viable protoplast remains unstained.
e) Fluorescein diacetate (FDA) staining: viable cells get stained by the FDA and can be detected by fluorescence microscopy.
5. Culture of Protoplast
Cell wall development around the protoplast membrane is the prior step of protoplast culture. The development of the cell wall is followed by cell division to form small colonies. Protoplast is generally culture in semi-solid Agar medium or Liquid medium.
Advantages of liquid culture over Solid culture
- It is easy to transfer
- Dilutions can be easily done in liquid culture
- Cell density can be easily manipulated
- Osmotic pressure can be easily changed in a liquid medium
Nutritional Requirements of Culture media
It is similar to those used in callus culture EXCEPT:
- The culture medium should not contain ammonium
- Very fewer quantities of iron and zinc are used
- The Calcium concentration is 2 to 4 times higher than the ordinary cell culture.
- Glucose is the preferred carbon source for protoplast culture
- A high Auxin/kinetin ratio is preferred to induce cell division
- A high kinetin/Auxin ratio is preferred for regeneration
Protoplast culture methods
- Feeder layer technique: in this technique the protoplast cell suspensions are exposed to x-rays, which inhibits cell division and metabolic activity followed by platting them on agar plates.
- Co-culture of protoplast: this technique is proffered when morphologically distinct protoplasts are used. So, generally, protoplasts of two different plant species can be co-cultured.
- Micro drop culture: In this technique, cuprak dishes, specially designed with the inner and outer chamber are used. The inner chamber contains a droplet of protoplast in the nutrition medium is added to wells. Whereas, the outer chamber is filled with water to maintain humidity. This technique has an advantage when there are few protoplasts in the droplet of the medium.
Regeneration of protoplast
Protoplast development can be done in two steps
- Cell wall formation: generally the process takes two to several days to develop cell wall in cultured protoplasts. The protoplast loses its spherical shape as the cell wall develops.
- Callus or Whole plant development: generally after cell wall formation, cell size increases, and cell division is initiated within 2-7 days. This cell division results in the formation of small colonies and visible colonies are formed by the third week. These colonies are developed in an osmotic free medium to form a callus. This callus may undergo several manipulations and undergo embryogenic or organogenic differentiation which results in the development of the whole plant.
Applications of protoplast culture and protoplast
- The whole plant can be generated by a culture of protoplast.
- Two protoplasts can be fused to form hybrids known as somatic hybridization
- Protoplast can be used for Single-cell cloning
- Chromosomes and cell organelles can be easily isolated from protoplast
- Transport and uptake studies or membrane studies can be easily done by protoplast
- Protoplast is used in genetic engineering for genetic transformations
- Ultrastructural studies can be done using protoplast
The millions of single cells are formed by protoplast isolation and protoplast culture, which can be used for wide varieties of studies
Multiple Choice Questions
1. Protoplasts are__________
a) Cell without a nucleus
b) Cell without a cell wall
c) Cell without plasma membrane
d) Cell without genetic material
2.__________ is the first step for protoplast culture
a) Selection of Explant
b) Viability testing for protoplast
c) Isolation of protoplast
d) Preparation of culture media
3. ___________ is the advantage of mechanical method for isolation of protoplast
a) The process is very tedious
b) Very less viable protoplast are isolated
d) Only B
4. Cellulase enzyme in isolation of protoplast is used ________
a) To degrade proteins
b) To degrade cellulose
c) To degrade pectin
d) To degrade hemicellulose
5. ____________ is used to check viability of protoplast
a) Phenosafranine staining
b) Fluorescein diacetate (FDA) staining
d) Only A
6. The following are the advantages of liquid protoplast culture EXCEPT:
a) It is easy to transfer
b) Dilutions can be easily done in liquid culture
c) Cell density can be easily manipulated
d) Less time consuming
7. Co-culture of protoplast is appropriate for ___________
a) Protoplast from two different organs of the same plant
b) Protoplast from the same organ of different plant
c) Protoplast from two different plant species
d) Protoplast from two same plant species
8. ___________media is used for protoplast culture
a) Synthetic media
b) Natural media
c) Nutritional media
d) None of the above
9. High Auxin/kinetin ratio in nutritional media for protoplast culture is preferred ______
a) To induce cell regeneration
b) To induce cell growth
c) To induce cell division
10. Advantage of Micro-drop protoplast culture technique is _____________
a) It requires Large numbers of protoplasts
b) It requires a Small number of protoplasts
c) It requires a large amount of culture media
d) It requires less amount of water
- Satyanarayana, biotechnology, 1st e.d. Kolkata, books & allied (p) ltd publishers, 2005 page:523-528