Protoplasts are isolated, single, and naked plant cells. Some
have the potential to (1) regenerate a cell wall, (2) dedifferentiate,
(3) divide mitotically and proliferate to form unlimited growing
cell clones, and (4) differentiate shoot and root meristems (or
embryos) which grow out to regenerate complete plants. Protoplasts
are potentially totipotent individuals at the single cell level.
Their freely accessible plasma membrane makes them ideal experimental
systems for many kinds of genetic manipulation.
Enzyme Solution (100 ml)
1.5% Cellulase ------------------------------------ 1.5 g
0.5% Pectinase ------------------------------------ 0.5 g
Distilled H2O -------------------------------------- 100 ml
Make fresh solution every time.
Regeneration Medium (1 liter)
MS Salts (Gibco) ----------------------------------- 4.3 g
B5 Vitamin Stock ---------------------------------- 1 ml
3% Sucrose ---------------------------------------- 30 g
0.6% Agar ------------------------------------------ 6 g
Add deionized H2O to make a final volume of ----- 1 liter
Adjust pH to 5.7 with NaOH.
B5 Vitamin Stock (100 ml)
myo-Inositol ---------------------------------------- 10 g
Thiamine-HCl --------------------------------------- 1 g
Nicotinic acid --------------------------------------- 0.1 g
Pyridoxine-HCl ------------------------------------- 0.1 g
1. Grow plants of a competent genotype of a herbaceous dicot in
potting compost in clay pots in a growth chamber (12 hour-light,
5000 lux, 27oC/20oC day/night temperature, 50/70% relative humidity) and water with
a 0.1%, commercial fertilizer solution at 8.00 am and 4.00 p.m.
2. Harvest leaves expanded to two-thirds their final size from
plants at the 7-9 leaf stage.
3. Wash leaves with tap water, and surface sterilize as follow:
Rinse in 70% ethanol three times.
Sterilize in 0.01% (w/v) Mercuric Chloride for 10 minutes.
Rinse leaves with sterile distilled water five times.
4. Arrange the leaves in stacks of 6, and carefully cut into fine
5. Transfer 500 mg sectioned leaves to 10 ml Enzyme Solution and
vacuum infiltrate to replace the intercellular air with Enzyme
6. Incubate at 28oC for 3 hours.
7. Observe the process of protoplasting from time to time under
the inverted microscope.
Gentle shaking helps to release protoplasts from the digested
tissue toward the end of the incubation period.
8. Filter the total digest through a 100-um stainless-steel sieve,
mixed with an equal volume of osmoticum (mannitol), and distribute
into capped centrifuge tubes.
9. Centrifuge for 5 minutes at 60g to sediment the protoplasts without sticking them too tightly
to the bottom of the tube.
10. Discard the supernatant by carefully pipetting, and resuspend
the sediment in the residual 0.5 ml of liquid, and fill the tube
with fresh osmoticum. Adjust the population density to 2 x 104/ml.
11. Pipet aliquots into petri dishes to give a thin liquid layer
which just covers the bottom.
12. Seal the dishes with parafilm and incubate at a constant temperature
of 26oC in the dark.
13. Cell wall regeneration and dedifferentiation are visible after
one day and the first divisions after three days. The division
frequency can be estimated 7-10 days after isolation by counting
dividing and non-dividing protoplasts in representative fields
and calculating the percentage of the total surviving population
which has divided at least once.
14. Dilute the suspension with 1/3 volume of culture medium with
reduced osmotic pressure and growth regulator concentration.
15. Repeat step 14 weekly and after a total of 4 weeks protoplast-derived
colonies become visible to the naked eye. When these have reached
a size of 1-2 mm in diameter, the plating efficiency (colony-forming
efficiency) is established by calculating how many proliferating
cell clones have developed per 100 protoplasts originally plated.
16. Transfer cell colonies onto the surface of agar-solidified
medium for further proliferation at low osmotic pressure.
17. After a total of 2 months the colonies are large enough (5
mm in diameter) to be transferred to Regeneration Media which promote the development of meristems and the outgrowth
of shoots and roots.
18. After a further month a fraction of the clones will have regenerated
multiple shoots (percentage regeneration efficiency) which are
rooted as cuttings on a medium with a low auxin concentration.
19. Two weeks later wash the rooted shoots carefully free of agar,
pot rooted shoots into a fine potting compost, and adjust slowly
to a low relative humidity. The plants can then grow further without
any special care.
Protoplast Preparation from Different Sources:
FACTORS AFFECTING PROTOPLAST PREPARATION AND REGENERATION
The ability of a cell to respond in specific ways to specific
stimuli during development. The competence on which the developmental
route from protoplast to plant depends describes the capacity
of specific plant cells to respond to isolation and in vitro conditions with a self-regulating programme of dedifferentiation,
proliferation, pattern formation, differentiation, and plant development.
2. Plant Species and Genotype
There are plant species, e.g., Nicotiana tabacum (Nicotiana, Solanaceae), where it has been relatively easy to establish conditions for
protoplast proliferation and plant regeneration from numerous
genotypes with protoplasts from different source tissues. There
are, however, also complete plant families, e.g., the Gramineae (grasses) where intensive efforts with hundreds of genotypes
of many species have never produced clear cut evidence for regeneration
of a single plant from a protoplast, and where even the induction
of protoplast division is still a severe problem.
3. Source Tissues from Organized Plants and from Cell Culture (A) Developmental State:
For plants grown in soil under natural conditions young plants
with expanding leaves before the differentiation of flowers generally
give best results. As soon as plants begin to flower, it is difficult,
if not impossible, to isolate proliferating protoplasts. The same
holds true for organs where senescence has started. One generally
uses organs before they have fully matured, e.g., expanding leaves
at one-half to two-thirds of their final size.
The protoplasts are generally easy to isolate from leaves and
to handle experimentally and it is usually no problem to isolate
very large populations of protoplasts at a similar state of differentiation
which will proliferate at a high plating efficiency and regenerate
These may provide a convenient source of proliferating protoplasts
in cases where attempts to culture protoplasts from leaves have
failed. Protoplasts from total seedlings, as well as from hypocotyl,
cotyledons, and from seedling roots have been successfully cultured.
These yield populations of tiny protoplasts as long as they are
immature. However, isolation of embryo is so tedious and culture
(E) Shoot Cultures:
Axenic shoot cultures proliferating under in vitro conditions are the source material of choice in species where
they can be easily established and maintained and many successful
protoplast laboratories are using them nearly exclusively.
(F) Cell Cultures: Organized cell cultures:Embryogenic cultures are becoming available in an increasing number
of plant species, including as interesting groups as the cereals
and legumes. The characteristic of these cultures is that small
groups of cell have the potential to differentiate to embryos.
(G) Cell lines:
A cell line culture type has arisen spontaneously in nearly every
plant species kept in culture for long periods of time, including
many cereals and grain legumes. Such cell lines are characterized
by a short cell cycle, proliferation on a single cell basis, friability,
and, unfortunately, a complete lack of morphogenic response.
4. Environmental Conditions (A) Care of plants:
The care of the plants before the isolation of protoplasts can
be a key factor. Plants which have been badly treated rarely yield
proliferating protoplast populations. Plants should be watered
at regular times with the correct amount of water or fertilizer
It is the experience that season can play a crucial role even
with optimized systems. This is not only experienced with greenhouse
material, where one can expect it, but also with material which
is grown in incubators and growth chambers without any direct
interaction with climatic factors. Experiments which do not work
between November and February may work well when repeated between
March and June.
Protoplasts isolated from plants grown under high light intensity
are very sensitive to the isolation procedures and, if they survive,
proliferate poorly. Experimental plants have to be kept in low
light intensity, i.e., 3,000 lux or lower, for at least 1 day,
and preferably longer, before the experiments.
(D) Temperature and Relative Humidity:
These contribute to the actual physiological state of the cell
at the time of isolation, but again there are no clear data to
aid the experimenter. In general it is advisable to grow the plants
under conditions close to their natural climate.
(E) Care of Cell Cultures:
Cell culture material can be very sensitive to alterations in
the culture conditions, e.g., the time interval of subculture,
the dilution ratio with fresh culture medium, the material and
geometry of the culture vessel, and the aeration and shaking conditions.
Isolation of protoplasts is often possible only during a short
time period in the exponential growth phase of the culture and
quite often cell cultures respond to unknown alterations in the
culture conditions in a way which prevents protoplast isolation
totally, or reduces their quality.
5. Endogenous Factors (A) Response to Wounding and Pest Treatments:
Mechanical injury can, within hours, alter the physiology of the
whole plant in such a way that protoplast culture is not possible
for several days. If leaves are taken from the same plant over
a long period of time, it is recommended that 1 week of recovery
be allowed between experiments. If pest treatments are necessary,
there should be a similar time interval between treatment and
isolation of protoplasts.
Excision of organs and preculture on a culture medium to induce
dedifferentiation in situ prior to protoplast isolation has been helpful with many recalcitrant
plants, e.g., legumes. On the other hand, attempts at a similar
approach with cereal leaves failed completely because the cell
walls were modified, in response to even 1 days preculture, such
that protoplast isolation was no longer possible.
(C) Cell Cycle Phase:
Fast cycling cells can be sensitive to the isolation procedure
but cell culture protoplasts are normally isolated from cycling
suspension cultures as opposed to resting ones. Protoplasts isolated
from fast growing cell suspensions rarely reach the high plating
efficiencies of arrested leaf mesophyll protoplasts, but this
may be due to other factors than cell cycle phase, such as the
state of differentiation.
6. Sterilization Procedure
The sterilization procedure has to be adapted to the type of organ
or tissue used in order to ensure that the majority of the cells
are not already killed or injured by the sterilizing agent. The
minimum concentration and the minimum time required for safe sterilization
has to be established for every case.
7. Mechanical Treatments Prior to Enzyme Application
The outer cell layer of plants has evolved to prevent access to
the inner cell layers. The cuticle, a complex layer of polymers
excreted to protect the cells, is not penetrable by the enzymes
used for protoplast isolation. The routine procedure is therefore
to mechanically bypass the epidermal cell layer by either peeling
off the epidermis (where possible), brushing with an abrasive,
or slicing the organ into cross sections. These methods require
some care and practice to avoid excessive damage of the inner
8. Plasmolysing Conditions
Plant cells have an internal pressure (turgor pressure) which
is contained by the rigid cell wall. Digestion of the wall in
a hypotonic environment would cause the protoplasts to burst.
Isolation is therefore carried out in hypertonic conditions which
leads to plasmolysis and frees the cell wall from its structural
9. Enzyme Treatment
Plant cell walls are complex chemical and physical structures
of celluloses, hemicelluloses, pectins, proteins, and other polymers.
The enzymes applied are mixtures of cellulases, hemicellulases,
and pectinases. The standard treatment is with a mixture of a
cellulose and a pectinase at concentrations around 0.5-2% w/v
dissolved in an osmoticum (giving 400-800 mOsm/kg H20) at pH 5.2-5.6 for a few hours. Enzyme concentrations higher
than 2% (w/v) are often deleterious although they reduce the time
10. Protoplast Harvest and Purification
Protoplasts are easily separated from incomplete digested tissues
by sieving through meshes with pore sizes adjusted to the size
of the protoplasts. Both washing and purification depend upon
differences in the relative buoyant density of protoplasts and
washing solution. Sugar and sugar alcohols (e.g., sucrose and
mannitol) have relatively high buoyant densities. Salt solutions
(e.g., CaCl2 and MgCl2) have relatively low buoyant densities at the same osmotic pressure.
Braun, AC, Wood, HN (1962) PNAS 48: 1776.
Cocking, EC (1960) Nature 187: 927.
Date, PJ (1983) in "Protoplasts 1983: Lecture Proceedings" (I.
Potrykus, C. T. Harms, A. Hinnen, R. Hutter, P. J. King, and R.
D. Shillito, eds.), p. 31. Birkhauser, Basel.