Protoplast Isolation and Culture
Unlike animals, in plants the plasma membrane is bound by rigid cellulosic wall, and adjacent
cells are cemented together by a pectin-rich matrix. It is mainly for this reason that somatic cell
genetics has advanced more for animals and than with plant systems.
- A means for genetic transformation of plants.
Essential ingredients of genetic modification of plants through protoplast system:
(i) Isolation of protoplasts
(ii) Culture of protoplasts to raise whole plants
(iii) Cell fusion
(iv) Introduction of genetic material into protoplasts
Isolation of Protoplasts:
- 1892 (Klercker): A mechanical method - The cells were kept on a suitable plasmolyticum
and cut with a fine knife. In this process some plasmolysed cells were cut only through
the cell wall, releasing intact protoplasts.
- 1960 (Cocking): Possibility of an enzymatic isolation – He used concentrated solution of
Cellulase enzyme to degrade cell wall.
- 1968 – Real progress with availability of both, cellulose and macerozymes.
- 1968 – the two enzymes were used sequentially on tobacco – first used macrozyme to
isolate single cells from meshophyll and then cellulose was applied to release protoplasts.
- 1968 (Power and Cocking): Two enzymes can be used together – one step process and
reduces chances of microbial contamination.
- Now a range of commercial preparations are available such as cellulosases,
hemicellulosase, pectinases etc.
- Protoplast isolations have been reported from mesophyll cells of in vivo and in vitro
grown plantlets, aseptic seedlings, microspores, embryogenic and non-embryogenic
suspension cultures.
Factors affecting yield and viability of protoplasts:
1. Source of material:
- Leaf is the most favourite sourec of plant protoplasts since it allows isolation of large
number of relatively uniform cells without necessarily killing the plants – mesophyll cells
are loosely packed and then enzymes have an easy access to the cell wall.
- When protoplasts are prepared from leaves the age of the plant and growth conditions of
the plant may be critical.
- For some plant species like cereals, isolation of protoplasts from the cultured cells is
easier.
2. Pre-enzyme treatments: (to facilitate access of the protoplasting enzymes)
- Cutinase to remove leaf epidermis
- Peel the lower epidermis and float the stripped pieces into enzyme solution
- Cutting leaf material into small strips and subject them to vacuum infiltration
- Brushing the leaf with soft brush or with the cutting edge of a scalpel may also improve
enzymatic action.
3. Enzyme treatment:
- The release of protoplasts is very much dependent on the nature and concentrations of
the enzyme used.
- Two enzymes are essential: (i) cellulose – degrades the cell wall (ii) pectinase – degrades
the middle lamella.
- Driselase has number of enzymatic activities: cellulose, pectinase, laminarinase,
xylanase.
- Spheroplasts: Some tissues require hemicellulase in addition to cellulose. A thin
cellulose-resistant layer would be left around the cells. E.g. aleurone cells of barley
needed additional treatment of glusulase to digest the remaining wall.
- Enzyme activities are also dependent on the pH and temperature.
4. Osmoticum:
- The protoplasts are osmotically fragile and they need to be protected using osmotic
stabilizers.
- Osmotic stabilizers should be present in the enzyme solution, the protoplast washing
medium and the protoplast culture medium etc.
- Protoplasts are more stable in a hypertonic solution rather than a isotonic solution; higher
level osmoticum may prevent bursting and budding.
- In solution of proper osmolarity the protoplasts appear completely spherical.
- Several carbohydrates such as glucose, fructose, galactose, sorbitol, mannitol proved
very effective solutes in adjusting osmotic pressure.
- CaCl2 in solution improves the osmotic stability of the plasma membrane.
Purification of protoplasts:
- After the enzymatic treatment the protoplasts are generated in the solution.
- In addition, there will also be the following in solution: undigested cells, broken
protoplasts, chloroplasts, vascular elements etc.
- Various steps of filtration and centrifugation helps to isolate the protoplasts.
Viability of protoplasts:
- It can be checked by different methods.
- Observation of cyclosis i.e. cytoplasmic streaming.
- Oxygen uptake measured by an oxygen electrode
- Photosynthetic activity
- Exclusion of Evan’s blue dye by intact membranes
- Staining with FDA
Protoplast Culture:
- Protoplasts can be cultured on solid, semi-solid and liquid media.
- Advantage of solid or semi-solid media is that the protoplasts remain remain stationery
and it is convenient to follow their development.
- However, liquid medium has been preferred because of the following reasons:
- (i) protoplasts of some species would not divide on agarified medium.
- (ii) The osmotic pressure of the medium can be effectively reduced after a few days
culture.
- (iii) If the degenerating component of the protoplast produces some toxic suabstances
which could kill the healthy cells it is possible to change the medium.
- (iv) The density of cells of special interest can be altered at will.
Cell wall formation:
- Within 2-4 days of culturing protoplasts loose their characteristic spherical shape and this
is taken as an indication of new cell wall regeneration.
- Cell wall regeneration can be demonstrated through staining with Calcofluor White and
microscopic techniques.
- There is a direct relationship between cell wall regeneration and cell division –
protoplasts which do not regenerate proper cell wall fail to undergo cell division – where
cell wall is poorly developed they show budding and several times enlargement of the
original volume – they may become multi-nuleate since karyokinesis is not followed by
cytokinesis.
Cell division:
- In protoplast cultures cell divisions are asynchronous – mitosis is normal. Several factors
influence cell division of protoplasts.
1. Nutritional requirements:
- Everything is required like mineral salts, sugars, organic aicds, vitamins, hormones etc.
- CaCl2 improved the percentage of dividing cells.
- Ammonium ion proved detrimental to survival of protoplasts.
- Hormones such as auxin/cytokinin are almost always required – the type of
auxin/cytokinin required and their ratios varies with the plant material.
- For cell division, actively growing culture requires high auxin/cytokinin ratio and highly
differentiated cells such as leaf cells require low auxin/cytokinin ratio.
- High level of ethylene correlates with low viability of protoplasts; addition of ethylene
inhibitors is effective.
- Anti-oxidants (glutathione, glutathione peroxidase, phospholipase) are essential to
counteract the phenolic browning of protoplasts.
2. Osmoticum:
- As discussed before.
3. Plating density:
- When grown in high densities, they tend to grow into each other and would result in the
formation of chimeric tissue if the protoplast population was genetically heterogeneous.
- When they are grown in low densities they become suitable for somatic hybridization and
mutagenesis etc.
4. Physical treatments:
- Electroporation treatment, high votage DC pulse (250-2000V) for µs, heat shock prior to
plating, chilling of freshly isolated protoplasts have been shown to trigger cell division
and plating efficiency.
5. Storage conditions:
- Freshly isolated protoplasts should be stored in diffused light or dark.
- For some species they are quite sensitive to light and should be stored in the dark.
- Protoplast cultures are generally maintained at 25-30°C.
6. Plant material:
- To ensure reproducibility in the protoplast culture, physiological state of the source tissue
is critical.
- The source tissue should always be taken from plants grown under controlled light, tempt
and relative humidity.
- One practice is maintaining axenic culture (a culture which is free from all other
contaminating organisms).
