Protoplasts are naked plant cells that have had their cell walls removed through enzymatic treatment. This document outlines methods for isolating and culturing protoplasts from plant tissues like leaves. Protoplasts are isolated using cell wall degrading enzymes like cellulase and pectinase, and stabilized in an osmotic solution like mannitol. Various culture techniques exist, including agar embedding and hanging drop cultures. Protoplasts can regenerate new cell walls and divide, or be fused with other protoplasts through techniques like PEG fusion to create somatic hybrids for applications like transferring disease resistance between plant species.

1. DR AKANKSHA JAIN

2. Synopsis:-
1.) Introduction
2.) History
3.) Isolation of protoplast
By mechanical method
By enzymatic method
4.) Methods of protoplast culture
Enzymes
osmoticum
5.) Purification of isolated
protoplast
By sedimentation and washing
Floatation
Other methods
6.) Protoplast viability testing and
density
7.) Culture media
For PC 1 group
For PC 2 group
For PC 3 group
Agar embedded culture
Micro chambers
HDC (hanging drop cultures)
MDA (Multidrop array)
Feeder layer
Co- culturing
Environmental factors
8.) Development of protoplast
Cell wall formation
Growth, division and plant
regeneration
9.) Fusion of protoplasts
10.) Verification and
characterization of somatic
hybrids
11.) Cybrid (somatic cell
hybridization or cytoplast)
12.) Conclusion
13.) References

3. Introduction
• Protoplasts are Naked plant
cells,
• All components of a plant
cell excluding the cell wall
􀂆
• Cell wall degrading enzymes
(cellulase) Used in cell fusion
studies, and to take up foreign
DNA, cell organelles, bacteria and
virus particles

4. • In 1880 - “Hanstein” introduced the
term protoplast to designate the
living matter, enclosed by plant cell
membrane.
• In 1960 – “cocking” first used the
enzyme to release the protoplasts.
He isolated the enzyme “cellulose”
from the culture of fungus i.e.
myrothecium verrucaria to degrade
the cell walls.
History

5. • Protoplast can be isolated
from the plant parts such as –
roots, leaves, fruits, tubers, root
nodules, endosperm, crown gall
tissues, pollen mother cells,
pollens and cells of callus
tissues grown in vitro.
Isolation of protoplast :-

6. 2 General
Methods :-
Mechanical
Enzymatic
Mechanical:-
• suitable for large
and highly
vacuolated cells,
such as onion bulb
scales
􀂆
• cells are
plasmolysed, tissue
is dissected and
deplasmolyzed

7. 2 General
Methods :-
Mechanical
Enzymatic
Enzymatic method:-
• commercial mixture of
cell wall degrading
enzymes in solution
containing osmotic
Stabilizers.
• But in some case the
enzymes have some
deleterious effects on
plant metabolism.

8. Technique used for isolation, culture and regeneration of plants from
leaf protoplast
Leaf sterilization
Epidermis
peeling
Peeled leaf segment
Plasmolysed cell in
enzyme mixture
Partial wall digestion
Pellets of
protoplasts
Isolated
protoplasts
Plating
of
protopla
st
Wall
regenerat
ion
First division
Clump of cells
Colony formation
Callus formation
Callus regeneration
Plant (in vitro)
Young plant

9. • Protoplast have been isolated from a variety of tissues and
organs including – leaves, petioles, shoot apices, roots, fruits,
coleoptiles, hypocotyls, stems, embryos, microspores, callus
and cell suspension cultures.
• A convenient and most suitable source of protoplasts is
mesophyll tissue from fully expanded leaves of young plants
and new shoots.
• Leaf tissue is popular because it allows the isolation of
large number of relatively uniform cells without the necessity
of killing the plants.
• In this method leaves are taken, sterilized and lower
epidermis from the excised leaves are peeled off. These are
cut into small pieces and then protoplasts are isolated.
Methods of protoplast culture:

10. Enzymes:-
• Enzyme Pectinase mainly degrades the
middle lamella.
• Cellulase and hemicellulase are required
for other main component
􀂆
• Helicase, colonase, cellulysin, glusulase,
zymolyase, pectolyase etc.
1. Two-step or sequential method
􀂆 pectinase (or macerozyme) → cellulase
2. One-step or simultaneous method
􀂆 one mixture of enzymes

11. Two step or sequential method:-
•The tissue is treated with a macro enzyme or pectinase enzyme
which separates the cells by degrading the middle lamella.
•These free cells are then treated with cellulose which releases the
protoplasts.
In general the cells are exposed to different enzymes for shorter
periods.
One step or simultaneous:-
The tissue is subjected to a mixture of enzymes in a one step
reaction which includes both macro enzyme and cellulose.
The one step method is generally used because it is less labor-
intensive.
During enzyme treatment, the protoplasts obtained need to be
stabilized because the mechanical barrier of the cell wall which
offered support has been broken.

12. • In isolating protoplasts, the wall pressure
that is mechanically supported by cell wall
must be replaced with an appropriate
osmotic pressure (also later in the culture
medium).
􀂆
• osmotic stress has harmful effects on cell
metabolism and growth: condensation of DNA in
cell nuclei and decreased protein synthesis
􀂆
• Lower osmotic potentials by addition of
various ionic and non-ionic solutes: mannitol,
sorbitol, glucose, fructose, galactose, NaCl, CaCl
etc.
Osmoticum:-

13. The mixture of the protoplasts is purified by a
combination of – filtration, centrifugation and
washing.
The enzyme solution containing the protoplasts is
filtered through the stainless steel or nylon
membrane to remove larger portion of undigested
tissues and cell clumps.
The filtered protoplasts – enzyme solution is mixed
with suitable vol of sucrose in protoplast suspension
medium to give final concentration and then the
centrifugation is done.
Ficoll and percoll are osmotically inert, their use may
be preferable over sucrose floatation.
Purification of isolated protoplasts:-

14. The most frequently used staining method for
processing protoplast viability are:
FDA is used i.e. fluorescein diacetate staining
method.
Calcofluor white (CFW) staining detect the
onset of cell wall formation.
Exclusions of Evans blue dye by intact
membranes.
Observation of cyclosis or protoplast steaming
as a measure of active metabolism.
Photosynthetic studies.
Protoplast viability and density:-

15. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
Murashige and
skoog medium:
for protoplast
culture
medium
supplemented
with 13%
mannitol
(W/V).

16. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
• Media calculated
under pc1 group with
minor changes.
• The changes are: the
concentration of
mannitol/ sorbitol
present in media
individually or in
combination reduced
by half.
• Concentration of
agar in the medium is
0.8%.

17. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
media described PC1
group with following minor
changes:-
(i.) mannitol/sorbitol/D-
glucose/D-ribose
individually or in
combination are omitted in
the media.
(ii.) The sucrose
concentration in all media
is adjusted to 2%.
(iii.) PC3 group media may
be prepared with 1.6% or
0.8% agar depending on
the mode of protoplast.

18. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
• The original method of
Nagata and Takebe for
the isolated protoplasts
is mixed with 1.0% agar
culture medium and
maintained at 40 to
45ْْ C.
• Small amount of agar
(liquid) protoplasts
mixture is then poured
into the sterile
petriplates

19. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
•A chamber constructed
of small rings with a
cover slip at the top.
•The joints of the
system are sealed with
the mineral oil.
•The simplest form is to
hold a droplet between
cover slips with a ring of
mineral oil around to
form a seal.

20. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
• Drop is placed at the
depression of a
specially prepared
hanging drop slide,
which is then under
sealed with a cover slip
and oil, or the drops are
placed on the lid of the
Petri dish.
• Petri dish contains
mannitol solution to
maintain the humidity.
The dish is sealed with
parafilm.

21. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
• This is a refinement
and amplification of
hanging drop
technique.
• Through this
technique becomes
easier to screen a
wide range of
nutritional and
hormonal factors
rapidly.

22. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
• A feeder layer consisting
of irradiated non-dividing
but living protoplasts
plated in agar medium on
Petri dishes.
• Protoplasts are placed on
this feeder layer at low
density a thin layer of agar
medium.
• This is especially
important when particular
mutant or hybrids are to be
selected on agar plates.

23. Culture media:-
1. For PC 1 group
2. For PC 2 group
3. For PC 3 group
4. Agar embedded
culture
5. Micro chambers
6. HDC (hanging drop
cultures)
7. MDA (Multidrop
array)
8. Feeder layer
9. Co- culturing
• It is the culturing of two
types of protoplasts- slow
growing and fast growing.
• Reliable growing protoplasts
preparation is mixed in vary
ratios with protoplasts of a
slow growing citrant species.
• The fast growing
protoplasts presumably
provide the other species with
growing factors and
undefined diffusible chemical
which aid the regeneration of
a cell wall and cell division.

24. Environmental factors:-
• It requires dim light for few days
then later on cultures are
transferred to the light of about
2000-3000 lux.
• Temperature required = 20-28
degree Celsius.
• PH = 5.5 to 5.9.

25. Protoplast Development:-
Regeneration of cell wall starts within a
few hours after isolation.
→ cell expansion
→ cell division (within 2-7 days)
→ cell colonies
→ callus is formed in an osmotic free
medium.
→ organogenesis/embryogenic
differentation

26. •􀂆 Mixing of protoplasts of two different genomes.
•􀂆 Spontaneous fusion: from callus tissue, do not
regenerate in to whole plants.
•􀂆 Induced fusion by
􀂆 polyethylene glycol method (PEG): high yield
􀂆 NaNO3 (sodium nitrate)
􀂆 Ca2+ at high pH
􀂆 electro fusion
•􀂆 3 main phases: agglutination or adhesion,
plasma membrane fusion at localized sites, fused
protoplasts
Protoplast fusion

27. Fusing protoplasts
Protoplasts aligning Fusion product

28. • It is a useful tool to make plant breeders
to make crosses between sexually
incompatible species for transfer of nuclear
or cytoplasmic characters.
• It is also used to make crosses:
within species (intraspecific),
between species (interspecific),
within genera (intra generic) and
between genera (intergeneric).
Use of protoplast fusion –

29. 􀂆 Morphological features (both vegetative and floral)
are usually intermediate between the two parents
•􀂆 not observed in dominant single gene traits such as
anthocyanin pigment, flower pigment, leaf size
􀂆
Isoenzyme analysis
•􀂆 unique banding patterns e.g. amylase, malate and lactate
dehydrogenase, esterase aspartate aminotransferase.
Chromosomal constitution
•􀂆 The chromosome number of the somatic hybrids should
be the sum of chromosome number of two parental
Protoplasts
•􀂆 However, variation is often observed (chromosome
number frequently higher).
Verification and characterization of
somatic hybrids

30. 􀂆 Molecular techniques:
• genetic constitution can be studied e.g. using
specific restriction patterns of chloroplast and
mitochondrial DNA, and molecular markers such
as
􀂆
AFLP (Amplified Fragment Length Polymorphism)
RFLP (Restriction Fragment Length Polymorphism)
RAPD (Random Amplified Polymorphic DNA)
micro satellites
Verification and characterization of
somatic hybrids

31. • In sexual hybrids, the cytoplasm is derived
from the maternal parent and in somatic
hybrids; it is derived from both the parents.
• However, hybrids can be obtained, where
nucleus is derived from one parent and
cytoplasm is derived from both, thus
producing cytoplasmic hybrids, which are
also called cybrids.
Cybrids:-

32. Fusion of protoplasts
Hybrid cells
Nuclear fusion No nuclear fusion
Complete hybrids Cytoplasmic hybrids

33. Applications:-
1.) Direct DNA uptake-
• for many sp., this was the only way to
transform cells before the particle gun.
• still useful for transient expression
studies.
2.) protoplast fusion to create somatic
hybrids
• "wide crosses" where even embryo
culture won't work
Citopsis gilletiana (wild) x Citrus sinensis
citrus sexually incompatible spp.
wild relative has disease/nematode resistance
somatic hybrid used as a rootstock

34. CONCLUSION

35. • Biotechnology –
By - S.S. Purohit
• Plant Biotechnology –
By – H.S. Chawla
• Biotechnology – Expanding Horizons –
By – B.D. Singh
• Biotechnology and Genomics –
By – P.K. Gupta
• Internet
References:-