protoplast

1. Plant Protoplasts
Biotechnology AdvancesVolume 23, Issue 2,
March 2005, Pages 131-171
Research review paper
Plant protoplasts: status and biotechnological perspectives
Michael R. Davey, Paul Anthony, J. Brian Power and Kenneth C. Lowe
School of Biosciences, University of Nottingham, UK

2. Molecular structure of the primary cell
wall in plants.

3. Plant cell wall
 Plant cell wall is a multilayered structure composed of
polysaccharides and proteins
 Polysaccharides are:
 Cellulose A polymer of glucose
 Pectic compounds Polymers of galacturonic acid
 Hemicellulose A polymer of a variety of sugars
e.g., xylose, arabinose and mannose
 Three types of layers constituting cell wall are:
 Middle lamella
 Primary wall
 Secondary wall
 found in plants, bacteria, fungi, algae,
 Absent from some archaea. Animals and protozoa
 Provide tensil strength and limited plasticity to cell
 Provide a tough physical barrier that protects interior of cell

4. Plant Cell Wall Functions
 strength to support the plant
 fix cell shape
 flexibility
 porosity
 water-proofing
 barrier to pests
 protection against environmental stress

5. Cell Wall

6. Cell tonicity

7. Protoplasts
 Protoplasts are spherical,
nacked bacterial, fungal or
plant cells obtained by the
removal of cell wall.
Protoplasts are
surrounded by plasma
membrane and potentially
capable of cell wall
regeneration, growth,
division and fusion etc

8. Plant Protoplast isolation methods
1. Mechanical isolation 2. Enzymatic isolation
1. Mechanical isolation (Klercker, 1892)
The cells are kept in a suitable plasmalyticum and protoplasts are
isolated by cutting the plasmolysed tissues with a sharp blade
through the cell wall when the tissue is again deplasmalysed.
This method is suitable for isolation of protoplasts from highly
vacuolated cells of stor age tissues like onion bulb, scales,
raddish roots etc.
Limitations
1) Low yield of protoplasts
2) Method is tedious and time consuming
3) Not useful for isolating protoplasts from meristematic tissues
4) Viability of protoplasts is low because of the presence of
substances released by damaged cells.
Advantages

9. 2. Enzymatic Isolation
(EC Cocking, 1960)
 Cocking, 1960 . A method for the isolation of plant protoplasts and vacuoles,
Nature 187 (1960), pp. 927–929.
 Turning point article plant protoplasts
Edward C. Cocking
In Vitro Cellular & Developmental Biology - Plant
Volume 36, Number 2 / March, 2000
Power and Cocking, 1970 . Isolation of leaf protoplasts: macromolecular uptake and
growth substance response, J. Exp. Bot. 21 (1970), pp. 64–70.

10. Enzyme Action
Major activity of cell wall degrading enzymes is in the digestion
of Pectins, Cellulase and Hemicellulases (xylans) that constitute the
plant cell wall.
Pectinases degrade the galacturonic acid residues of pectins that
confer the cell to cell adhesion, and apparantly macerate the tissue
to single cells. (Macerozyme; Rhizopus spp.)
Cellulases digest the cellulose component, conferring the
spherical shape to the protoplasts (Cellulase R10; Trichoderma)
Hemicellulases assist in the breakdown of xylans (Rhozyme HP
150)
Driselase (cellulytic and proteolytic activity)

11. Enzyme types for protoplast isolation
 Plant cells Cellulase, pectinase, xylanase
 Gram-positive Bacteria Lysozyme (+EDTA)
 Fungal cells Chitinase
Methods for Enzymatic Isolation of Protoplasts
 Sequential or Two-step method (Takabe et al., 1968)
 Simultaneous or One step method: (Power and Cocking, 1968)

12. Advantages of Enzymatic Isolation of Protoplasts
1) large scale reproducible isolation of protoplasts from
various tissues and more or less universal application
2) osmotic shrinkage is minimum and the deleterious effects
of excessive plasmolysis are minimized
3) cells remain intact and are not injured as is the case of
mechanical methods of isolation
4) protoplasts are readily obtained

13. Factors affecting protoplast isolation
 Physiological state of tissue and cell
material
 Temperature
 duration of enzyme incubation
 pH of the enzyme solution
 gentle agitation
 and nature of the osmoticum

14. Conditions required for enzyme activity
• Enzymes are pH and temperature dependent, thus,
for enzymatic release of protoplast an enzyme
showing activity at pH range 4.7-6.0 and temperature
range of 25-30°C is used
• Duration of enzyme pretreatment and condition of
light presence required for incubation may also be
determined.
• Enzyme mixture used should essentially consist of
cellulose, hemicellulase and pectinase which facilitate
the degradation of cellulose, hemicelluloses and
pectin, respectively.
• The concentration of sugar alcohols used as
osmoticum (mannitol) must be empirically defined.

15. Source tissues for protoplast isolation
 Cell suspension
 Callus
 Leaf
 Root nodules
 Guard cells
 Stems, embryos, microspores, shoot apices,
petioles, hyocotyles etc

16. Source material
Leaves:
 Uniform ppts w/o killing plant
 Cells are loosely arranged, making enzyme
access easy
 Cereal leaves not good ppt source
Cell suspensions
Embryogenic, frequently subcultured and early log
phase

19. Protoplast isolation steps
 Selection of source material
 Sterilization of the tissue
 Preparation, filteration of enzymes
 Incubation of the tissue with the enzyme solution
 Release of protoplasts
 Filteration of protoplast solution through an autoclaved
mesh to remove undigested tissues
 Repeated washing by centrifugation to remove enzymes
 Separation of protoplasts from debris
Since removal of the cell wall results in loss of wall pressure upon the cell,
protoplasts are isolated and maintained in hypertonic plasmolytica provided by
a balanced inorganic salt medium or monosaccharide sugar solution. Mannitol,
for example, is not readily transported across the plasmalemma and therefore
provides a stable osmotic environment for the protoplast.

20. Arabidopsis leaf ppt isolation

21. Brachypodium ppts

24. Pre-Requisites of Protoplast Culture
 Determine the Yield of protoplasts
 Counting is essntial to adjust suitable plating density (final
density) for various objectives (Culture, transformation,
electroporation )
 Haemocytometer marking with chamber depth of 0.2 mm and
vol. of each small sub-unit of 1/16 mm is used
Calculate the yield as :
Y= n x 103 x X
n = No. of protoplasts counted in 5 triple-ruled squares
X = Volume of protoplasts in suspension
Adjust the protoplast suspension to desired plating density
 Determine the Viability of protoplasts
viability is determined by staining with Flourescein Diacetate
(FDA), Evans blue, Phenosafranin etc.

25. Wheat protoplasts
Haemocytometer

26. Automated Cell Counter
Load the sample onto a slide.
Insert the slide into the TC20 cell counter;
counting automatically begins.
Obtain a total cell count (without trypan
blue) or total and live cell counts (with
trypan blue) in 30 seconds.

27. Protoplast Viability stimation

28.  Two methods were evaluated in order to assess the viability of
protoplasts and cell suspensions of Coffea arabica cv. Catimor used in
a protocol of transformation by electroporation. One method consisted
of staining with 1 % Evans blue and the other staining with 1% 3-[4,5-
dimethyltiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). When
Evans blue was applied to viable cells and protoplasts they either did
not stain or acquired a faint blue colour. However, it was difficult to
distinguish the non-viable cells in the wide spectrum of clear blue
tonalities. In contrast, with the MTT assay only the viable cells and
protoplasts reduced this salt to the red coloured formazan; viable and
non-viable cells were distinguished more clearly with MTT than with
Evans blue. The optimal temperature for the reaction with MTT was
37ºC. The time of incubation was shown to be important, since longer
times improved the reaction; the highest viability value was obtained
after incubation for 120 min
 1% 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT).

29. Protoplast Culture
 Most culture media are MS (Murashige & Skoog, 1962) and B5 (Gamborg et al,
1968)-based with the addition of an osmoticum (nonmetabolizing sugar alcohol
such as mannitol or somewhat soluble, sorbitol)
 Ammonium salts have been found detrimental to protoplasts survival of many
species, preferred media have reduced concentration or no ammonium.
 Concentration of zinc is reduced while the concentration of calcium is increased
as it enhances the membrane stability.
 Complex medium with coconut milk (Kao & Michayluk, 1975) used for the
culture of protoplasts at very low densities
 Major growth regulators auxins and cytokinins are normally essential for
sustained protoplast growth
 Some exceptions are requirement of only auxins e.g., in carrot and A. thaliana.
 Auxins and cytokinins are detrimental to the growth of citrus protoplasts
 Agarose as solidifying agent gives better results than agar
 Sucrose and glucose are regular choices of carbon sources in most media, in
cereals a change in carbon source from sucrose to maltose promotes
regeneration.
 Environmental conditions: High light intensity inhibits growth of protoplast
hence initially protoplast is grown in dim light for few days and then transferred
to light of about 2000-5000 lux. However, better results are obtained when
cultured in darkness.

30. IPE: (Calculated 15 d after culture)
The percentage of originally plated protoplasts regenerating
a new cell wall and which had undergone one or more
mitotic divisions.
FPE: (Calculated 30 d after culture)
The percentage of original protoplasts that had given cell
colonies consisting of more than twenty cells.

31. Osmoticum
During isolation and culture, protoplasts require osmotic protection until they
regenerate a strong wall. Inclusion of an osmoticum in both isolation and culture
media prevents rupture of protoplasts. A variety of ionic as well as non-ionic solutes
have been tested for adjusting the osmotic potential of various solutions used in
protoplast isolation and culture. The most widely used osmotica in a protoplast
culture medium as well as in an enzyme mixture are sorbitol, mannitol, glucose, or
sucrose. Protoplasts are more stable in a slightly hypertonic solution. The osmolarity
of the medium is gradually reduced by periodic addition of a few drops of fresh
medium as soon as the protoplasts have regenerated walls and under- gone divisions.
Ionic substances (335 mmolL-1 KCI and 40 mmolL-1 MgS04.7H2O) improve the
viability of protoplasts and yield cleaner preparations. Usually enzyme solutions are
supple- mented with certain salts (5-100 mmolL-1 CaCl2) along with non-ionic
osmotic stabilisers. Cocking and Peberdy (1974) developed a cell-protoplast washing
(CPW) solution containing salts and a suitable osmoticum to provide stable and
cleaner preparations. The CPW solution can be used during enzyme incubation and
washing of protoplasts. The enzyme incubation period depends on its concentration
in the solution and the type of material used.

32. Plating density
Like cell cultures, the initial plating density of
protoplasts has profound effect on plating efficiency.
Protoplasts are cultured at a density of 1 x 104 to 1 x 105
protoplasts ml-1 of the medium.
At high density the cell colonies arising from individual
protoplasts tend to grow into each other resulting into
chimera tissue if the the protoplast population is
genetically heterogeneous.
Cloning of individuals cells, which is desirable in
somatic hybridization and mutagenic studies, can be
achieved if protoplasts or cells derived from them can
be cultured at a low density.

33. Protoplast culture methods
1) Liquid layers
2) Embedded in agarose/agar
3) Liquid over agarose
4) Alginate encapsulation
5) Hanging/sitting droplet culture
6) Filter paper substratum placed on agar
7) Microdrop array technique

34. Protoplast Droplet Culture

35. Protoplast Development and Regeneration
Protoplast starts to regenerate a cell wall within few days (2-4 days) of
culture and during this process, protoplasts lose their characteristic
spherical shape. Cell wall regeneration can be confirmed by Calcofluor
White staining method.
There is direct relationship between wall formation and cell division.
Protoplasts with a poorly developed wall often show budding and may
enlarge several times their original volume. They may become
multinucleate because karyokinesis is not accompanied by cytokinesis.
Among other reasons, inadequate washing of the protoplasts prior to
culture leads to these abnormalities.
Once the cell wall formation is completed, cells undergo division resulting in
increase size of cells. After an interval of 3 weeks, small cell colonies
appear, these colonies are transferred to an osmotic-free callus induction
medium. This is followed by introduction into organogenic or embryogenic
medium leading to plantlet development.

36. Maintenance of Protoplast Cultures
 Incubate cultures for 2 wks in dark at 25-30°C depending upon
the crop species
 Afterwards transfer to continuous illumination from 1000 lux to
3000 lux.
 Cell wall formation is evident by the change of protoplast shape
and by Calcofluor white staining
 Division starts by 3-7 d of culture initiation
 Sequential dilution of osmoticum of the culture medium
 Once callus formation is achieved plant regeneration process is
the same as of regular tissue culture technique.

37. Protoplast-to-plant regeneration

38. Exploitation of Protoplast-to-plant Technologies
 Somatic hybridization to generate novel plants
 Transformation of protoplasts
 DNA uptake into the nucleus of isolated protoplasts
 Organelle transformation
 Molecular Farming
 Somaclonal variation

39.  Protoplasts may be produced, under aseptic conditions, from a wide range
of plant species either directly from the whole plant, or indirectly from in
vitro cultured tissues.
 From a physiological viewpoint, however, the protoplast cannot be
regarded simply as a cell lacking a wall, since the mechanics of isolation,
in conjunction with environmental factors, undoubtedly influence its
metabolism and elicit subtle ultrastructural changes. The absence of a
functional cell wall may affect the permeability of the cell membrane and
lead to a general leakage of solutes from the protoplast.
 The protoplast is also in a transient state since most protoplasts,
irrespective of their immediate cultural environment, will initiate the
synthesis of a new cell wall a few hours after release, and eventually revert
to a single-walled cell.
 In spite of these considerations, protoplasts provide an important
biochemical tool for the biologist.
 In the absence of a cell wall, the exposed plasmalemma can be examined
in great detail with respect to particle uptake, permeability, possible
membrane-associated functions such as disease resistance, membrane
fusion, and, during cell wall synthesis, the relationship of the
plasmalemma to its cell wall.

40.  Details of slide # 3
Middle lamella The first layer formed during cell division, forming the outer surface
of the wall and is shared by adjacent cells. It is composed of pectic compounds
and proteins
The primary wall: Formed after the middle lamella consisting of a framework of
cellulose microfibrils embedded in a gel-like matrix of pectic compounds,
hemicellulose and glycoproteins.
Secondary wall is formed after cell anlargement, a rigid structure composed of
cellulose, hemicellulase and lignin.
Cell wall fulfills several important roles but two main fnctions are
1. To provide tensil strength and limited plasticity so that the cell can develop high
turgor pressures without rupturing. Turger pressure provides support for non-
woody plants.
2. Provide a tough physical barrier tat protects the interior of cell from incvading
microorganisms.
The properties of plant cell wall that make it an efficient protective barrier restrict the
use of plant cells for a variety of cell and tissue culture techniques, including the
delivery of molecules into the cell, somatic hybridization and genetic manipulation.
These techniques can only be applied to plant cells after removal of cell wall.

41. When large populations of the protoplasts are required which is the
norm, enzymatic digestion of cell wall is essential. Interestingly it was
the release of protoplasts by natural degradation of cell wall during
fruit ripening that stimulated investigations more than 4 decades ago
of protoplast isolation from roots of tomato seedlings by E. C.
COCKING (1960, University of Nottingham).
Protoplast preparation was at first done in two steps:
At first was the middle lamella dissolved by pectinases,
then was the cell wall broken down by cellulase.
The process is further simplified by a mixture of enzymes
The enzymes required for this procedure are usually not pure, but
crude extracts from certain bacteria and fungi. Nowadays is the
method often shortened to just one operation in which a mixture of
enzymes is applied.

42. Details of slide no. 10 Determine the yield of protoplasts
Counting is essntial to adjust suitable protoplast plating density (final density) for
various objectives (Culture, transformation, electroporation ). Plating density of
protoplasts in the culture medium is crucial for maximising wall regeneration and
concomitant daughter cell formation. Generally plating density is in the ranges 5 x 10
to 1 x 106 protoplasts per ml. An exessive high p.density rapidly depletes nutrients an
protoplast-derived cells can fail to undergo sustained division Cells stimulate mitotic
division of adjacent cells by releasing growth factors including amino acids into the
surrounding medium, a process known as medium conditioning or nurse culture.
Consequently, when protoplasts are cultured below the minimum inoculum density
threshold they don’t survive as there is no medium conditioning.
Haemocytometer marking with chamber depth of 0.2 mm and vol. of
each small sub-unit of 1/16 mm is used for counting protoplasts.
i. Isolated protoplasts adjusted in final vol. of 10 ml of medium
ii Moisten edges of haemocytometer and place cover slip firmly
iii. Add a drop of well-mixed protoplast suspension to the counting chamb
of H. cytom.
iv. Count the no. of protoplasts in 5 triple-ruled squares each with 16 sigle
ruled small squares v. Calculate the yield :
Y= n x 103 x X
n = No. of protoplasts counted in 5 triple-ruled squares
X = Volume of protoplasts in suspension
vi. Adjust the protoplast suspension to desired plating density

43. FDA is relatively nonpolar and passes freely across the
plasma membrane. In association with viable
protoplasts/cells, the molecules are hydrolyzed by the
action of esterases to release polar fluorescein molecules
which can not cross the membrane and consequently
accumulate in the cell and stain it bright green under UV
illumination. Thus, metabolically active protoplasts are
visualized. Non-viable protoplasts appear orange red.
Fluorescence Phase Contrast microscope is required.

44.  Slide # 11 Isolated protoplasts commence cell wall regeneration within a short time (often
minutes) following introduction into culture. However, they require osmotic protection until
their primary walls can counteract the turgor pressure exerted by the cytoplasm. In some cases,
gradual reduction of osmotic pressure by diluting the culture medium with a solution of similar
composition but of reduced osmotic pressure is essential for sustaining mitotic division,
leading to formation of daughter cells and tissues. Protoplasts from different species and from
different tissues of the same species may vary in their nutritional requirements.
 Many culture media have been based on MS (Murashige & Skoog, 1962) and B5 (Gamborg et
al, 1968) formulations with the addition of an osmoticum, usually a nonmetabolisable sugar
alcohol such as mannitolor somewhat soluble, sorbitol. Ideally media should be simple and
fully defined to ensure reproducibility b/w labs.
 An exception is the complex, undefined medium containing coconut milk (Kao & Michayluk,
1975) used for the culture of protoplasts at very low densities.
Major growth regulators auxins and cytokinins are normally essential for sustained protoplast
growth ,some exceptions are requirement of only auxins e.g., in carrot and A. thaliana.
Auxins and cytokinins are detrimental to the growth of citrus
Growth requirement of protoplasts often change during culture, necessitating modification of
Medium composition, typically involving reduction of the auxin conc.
Sucrose and glucose are regular choices of carbon sources in most media, in cereals a change
in carbon source from sucrose to maltose promoted regeneration.
Several approaches are developed for protoplast culture all of which are based on liquid or
semi-solid media, or their combination Protoplasts are plated by one of the methods: 1)Liquid
layers. 2) Embedded in agarose/agar. 3) Liquid over agarose 4) Alginate encapsulation 5)
Hanging/sitting droplet culture. 6) Filter paper substratum placed on agar. 7) Microdrop array
technique
Agarose has been used in place of gar
Maintenance of protoplast culture

45. # 15. Protoplast-to-plant technologies are being exploited in different areas of plant cell research.
Somatic hybridization to generate novel plants: A no. of recent papers have describes the generation of unique
plants through somatic hybridization by protoplast fusion.Citrus, Brassica, Potato and other members of
Solanaceae, cereals, ornamentals and miscellaneous crop plants have been targetted for the incorporation of useful
traits via this technique.
 Transformation of protoplasts:
 DNA uptake into the nucleus of isolated protoplasts
 Because the plasma membrane has fluid mosaic characteristics, DNA uptake can be induced by chemical /or
physical procedures.Treatment of protoplast-plasmid mixtures with PEG or Electroporation is the approach
normally exploited to induce DNA uptake into protoplasts. However, transformation frequencies remain low,
owing to the reproducible protoplast-to-plant system. PPT can be cotransformed with more than one gene carried
on the same or separate plasmids. This strategfy is especially important in transforming plants that are not
amenable to other methods of gene delivery.
 Organelle transformation
The advantage of plastid transformation over nuclear transformation include high foreign protein synthesis in
these organelles and the absence of epigenetic effects in regenerated plants. IOne key attraction of
chloroplast engineering is the apparent lack of transgene transmission through pollen. The recent success in
plastid transformation gives hopes of successful future transformation of mitochondria as well.
 Molecular Farming: It is transformation for the expression of recombinant proteins. In the recent
years there has been growing interest in the use of plants as expression systems for the production of recombinant
proteins. At present several plant-derived pharmaceutical proteins are at an advanced stage of commercial
development, such as antibiotics, vaccines, human blood products, hormones and growth regulators
 Somaclonal variation somatic hybridization and transformation technologies provide reliable
approaches for combining interspecific and intergeneric traits and targeted modification of genomes, however,
somaclonal variation or protoclonal variation may involve expression and release of naturally occuring genetic
variation as a result of culture and may be regarded as a simple form of genetic manipulation. Protoclonal
variation is more evident in the form of alterations in morphological characters of the plants. The attraction os
somaclonal or protoclonal variation is that it requires no knowledge of the genetic basis of specific traits, it
negates DNA recombinant techniques, it does not require mutagenic agents, specialized apparatus and it can be
exploited through routine culture procedures.