Protoplast Culture

1. Protoplast culture

2. Presented by;
Samra Hafeez

3. Modules
Introduction
Historical development
Protoplast isolation & Methods
Protoplast purification & viability test
Protoplast fusion & Techniques
Protoplast culture
Culture methods
Regeneration of protoplasts
Applications
Advantages & Disadvantages
Summary

4. What is protoplast?
 The protoplasm of a living plant or bacterial cell
whose cell wall has been removed.
 Protoplasts are plant cells that have been stripped
of their cell walls through the action of pectinases
and cellulases.
In
case
of
Plants

5. HISTORICAL DEVELPOMENT

6. PROTOPLAST ISOLATION
Protoplasts are isolated by two techniques
 The essential step of protoplast isolation is the proper use
of Osmoticum.
Mechanical Method
Enzymatic Method

7. Sources of Explant for Protoplast Isolation
 Protoplasts can be isolated directly from the
different parts of whole plant which bears the soft
parenchymatous tissue (e.g., young fully expanded
soft leaves) or indirectly from the in vitro grown
plant tissue (e.g., callus tissue).
 Before isolation of protoplast, the source material
if it is from in vivo grown plant then it should be
properly surface sterilized using the proper method
of sterilization. Then any of the methods either
mechanical or enzymatic can be used to isolate the
protoplast

8. Mechanical Method
Any soft parenchymatous tissue is kept in a plasmolyticum.
The plasmolyzed tissue is then finely chopped into pieces and the intact
cells (plasmolyzed) are released into the medium from the cut surface.
The suspension is then allowed for deplasmolysis and the released
protoplasts attain their original size.

9. Mechanical Method

10. Limitations Of Mechanical method
 Yield of protoplasts and their viability is low.
 It is restricted to certain tissues with vacuolated cells.
 The method is laborious and tedious

11. Enzymatic Method
Young fully expanded soft leaves, or in vitro grown callus tissue or
cell suspension culture grown cells can be used as the source
material.
The tissues or cells are incubated in plasmolyticum for 1 hr.
before enzymatic treatment.
The intact tissue materials cut into smaller pieces to increase the
surface area of enzymatic activity.
The enzymes can be used either sequentially in two step method
or in a single step by mixed enzymatic method.

12. Enzymatic Method

13. Enzymes
 The enzymes used are of three main categories:
 Cellulase
 Hemicellulase
 Pectinase
 The concentration of enzymes used and the time period of
incubation varies greatly depending on the tissue type

14. Advantages of enzymatic method
 Used for variety of tissue and organs such as fruits, roots,
petioles and leaves
 Osmotic shrinkage is minimum
 Cells remain intact and not injured
 Protoplast readily obtained

15. Purification of protoplasts
 For purification, the protoplasts suspended in osmoticum
are centrifuged using sucrose (20%) solution.
 The viable protoplasts float on the top surface of sucrose
solution forming a band.
 These protoplasts are then collected, re-suspended in
osmoticum and washed several times.
 counting the number with the help of hemocytometer

16. Protoplast Viability Test
1. Fluorescein
diacetate (FDA)
dissolved in
acetone is used
at a conc. of
0.01% and intact
viable protoplasts
only fluoresce
when observed
under UV.
2. Phenosafranine
is also used at a
conc. of 0.01 %,
which is specific
for dead
protoplast that
shows red in
color.

17. Protoplast fusion
 Somatic fusion, also called
protoplast fusion, is a type
of genetic modification in
plants by which two distinct
species of plants are fused
together to form a new
hybrid plant with the
characteristics of both, a
somatic hybrid.

18. Protoplast Fusion techniques
 Electrofusion
 Polyethylene glycol induced fusion(PEG)
 High Ca+2 ,High PH

19. ELECTROFUSION
 Mild electro stimulation
 Two glass capillary
microelectrodes
 An electric field of low strength
 Leads to pearl chain
arrangement of protoplast
 Application of high intensity
electric impulse for some
microseconds
 Breakdown of membrane and
subsequent fusion

20. peg
 This chemical has been
identified as a possible fusogen
 Has a molecular weight of
about 1500-6000
 Usually PEG solution of about
28-50% is used
 This polymer binds to the lipid
membrane of the cell and thus
induces fusion
 Fusion takes place for 45 min in
incubation

21. PEG RESULS OF CLASS PRACTICAL

22. Protoplast culture
 Isolated protoplast can be cultured in an appropriate medium to
reform cell wall and generate callus
Protoplasts are cultured either in
 Agar medium
 Liquid medium

23. AGaR culture
 Agarose is the most frequently used agar to solidify the culture
media.
 The concentration of the agar should be such that it forms a soft
agar gel when mixed with the protoplast suspension.
 In agar cultures, the protoplasts remain in a fixed position, divide
and form cell clones.
 The advantage with agar culture is that clumping of protoplasts is
avoided.

24. LIQUID CULTURE
 Liquid culture is the preferred method for protoplast cultivation for
the following reasons:
 It is easy to dilute and transfer.
 Density of the cells can be manipulated as desired
 For some plant species, the cells cannot divide in agar medium,
therefore liquid medium is the only choice.
 Osmotic pressure of liquid medium can be altered as desired.

25. Protoplast culture
 Protoplast cultured in suitable nutrient media first generate a new
cell wall
 The formation of a complete cell with a wall is followed by an
increase in size, number of cell organelles and incubation of the
cell division
 The first cell division may occur within 2 to 7 days of culture
 Resulting in small clumps of cells, also known as micro colony
with in 1 to 3 weeks
 From such clumps, there are 2 ways to generate a complete plant
 Through organogenesis
 Osmatic embryo converted into whole plant through germination

26. Culture Methods
Micro drop culture
Co-culture of protoplasts
Feeder layer technique
The culture techniques of
protoplasts are almost the
same that are used for cell
culture with suitable
modifications.

27. FEEDER LAYER TECHNIQUE
For culture of
protoplasts at low
density feeder layer
technique is
preferred.
This method is also
important for
selection of specific
mutant or hybrid cells
on plates.
The technique
consists of exposing
protoplast cell
suspensions to X-
rays (to inhibit cell
division with good
metabolic activity)
and then plating them
on agar plates.

28. Co-culture of protoplasts
Protoplasts of two
different plant
species (one slow
growing and another
fast growing) can be
co- cultured.
This type of culture is
advantageous since the
growing species provide
the growth factors and
other chemicals which
help in the generation of
cell wall and cell
division.
The co-culture
method is generally
used if the two types
of protoplasts are
morphologically
distinct.

29. Micro drop culture
Specially designed
dishes namely
cuprak dishes with
outer and inner
chambers are used
for micro drop
culture.
The inner chamber
carries several wells
wherein the
individual
protoplasts in
droplets of nutrient
medium can be
added.
The outer chamber
is filled with water
to maintain
humidity. This
method allows the
culture of fewer
protoplasts for
droplet of the
medium.

30. Regeneration of Protoplasts
 Protoplast regeneration which may also be regarded as protoplast
development occurs in two stages:
1. Formation of cell wall
The process of cell wall formation in cultured protoplasts starts
within a few hours after isolation that may take two to several days
under suitable conditions. As the cell wall development occurs, the
protoplasts lose their characteristic spherical shape. The newly
developed cell wall by protoplasts can be identified by using
calcofluor white fluorescent stain.

31. Regeneration of Protoplasts
2. Development of callus/whole plant
 As the cell wall formation around protoplasts is complete, the cells
increase in size, and the first division generally occurs within 2-7
days. Subsequent divisions result in small colonies, and by the end
of third week, visible colonies (macroscopic colonies) are formed.
These colonies are then transferred to an osmotic-free (mannitol or
sorbitol-free) medium for further development to form callus.
 With induction and appropriate manipulations, the callus can
undergo organogenic or embryo genic differentiation to finally form
the whole plant.

32. Applications
STUDY OF OSMOTIC BEHAVIOUR
STUDY OF IAA action
Study of cell wall formation
Organelle isolation
Study of morphogenesis
Virus uptake and replication
Gene transfer
Induction of Mutation and Genetic Variability
Implantation of Chloroplast
Transplantation of Nuclei
Transplantation of Chromosome
Somatic Hybridization

33. Study of Osmotic Behavior
 Influence of different
environmental factors on
the osmotic behavior can
be studied using plant
protoplasts.

34. Study of IAA Action
When growth promoters like IAA are
applied to plants, they act directly
on plasma membrane of the cell and
increase the permeability of the
membrane to water resulting in cell
elongation. This can be established
by the use of protoplast in vitro.
When IAA is applied to the
plasmolyticum containing
protoplasts they expand rapidly and
finally burst due to too much
vacuolation. Further, it can be
verified by using anti-auxins that
suppress this bursting, indicating
that the site of action of IAA is the
plasma-lemma of the plant cell.

35. Study of Cell Wall Formation
 The early deposition of
cellulosic micro-fibril and
their orientation at the
protoplast surface can be
followed using both light and
electron microscope and
has also provided much
basic information
concerning cell wall biology.

36. Organelle Isolation
 Protoplasts are very convenient material for the isolation of
chloroplasts, mitochondria, nuclei and even chromosomes. It has
been demonstrated that chloroplasts particularly isolated from
cereal protoplast have higher capacity for CO2 fixation than those
obtained by mechanical grinding.

37. Study of Morphogenesis
 Isolated protoplast provides an ideal single cell system. Under
suitable condition, protoplast regenerates its own wall and
become the walled cells. Cell division followed by plant
regeneration may occur from such unique single cell system either
through organogenesis or embryogenesis.

38. Virus Uptake and Replication
But after the innovation of protoplast isolation and its culture, this problem is
almost solved. Protoplast can directly be inoculated with pathogenic virus in
the medium. The process of uptake of virus particle, their replication inside
the protoplasts and their mode of action at the molecular and cellular level
are made possible by the aid of protoplasts.
The plant virus interrelationships in the past were not clearly known due to
lack of suitable experimental systems that can easily infect the cells.

39. Isolation of Bacteroides from Root Nodule Protoplast:
 Viable Bacteroides from root nodules of legumes has been
isolated by first preparing nodule protoplast and then rupturing
them either mechanically or by lowering suddenly the concen-
tration of the plasmolyticum in the surrounding medium. This
method ensures the freedom of the preparation of bacteria from
the infection thread.

40. Induction of Mutation and Genetic Variability:
 It has been repeatedly observed that plant cell in culture show a
wide range of genetic diversity. This phenomena can be exploited
by plant breeders and geneticists for inducing variability in
protoplast culture. The recessive characters can be detected in
the regenerated plants derived from haploid protoplasts.
Therefore, haploid protoplast would make an ideal system for
studying the effect of irradiation and for the induction of mutation
by plating them in media supplemented with various chemical
mutagens.
 From this method, mutant line can be selected.

41. Implantation of Chloroplast
 Plant protoplasts have ability to
uptake the isolated chloroplasts
by the process of endocytosis.
Several reports have described
uptake of chloroplasts.
Chloroplasts isolated from
Vaucheria dichotoma were
implanted into carrot cell
culture protoplasts. The
chloroplasts may enter the
cytoplasm enclosed in
membrane-bound vesicles,
although the enclosing
membrane in some cases is
absent

42. Transplantation of Nuclei:
 Isolated nuclei can be introduced into the protoplasts. Both intra
and inter-specific nuclear transplantation have been observed in
Petunia hybrida, Nicotiana tabacum and Zea mays. Retention,
normal function or degradation of the incorporated nuclei is not
known. But it is really opening up new avenues for the study of
nuclear- cytoplasmic interaction if fertile plants with foreign
nuclei could be regenerated from such protoplasts.

43. Transplantation of Chromosome
 The uptake of isolated metaphase chromosomes has proven
successful in plant protoplast. This procedure provides a valuable
method for genetic information transfer and gene analysis

44. Somatic Hybridization:
 Fusion of protoplast that facilitates the mixing of 2 whole
genomes and could be exploited in crosses at
 Intergenic,intekingdom and interspecific level
 Somatic hybridization is used to produce hybrids from sexually
incompatible species
 This method could also be used to produce selection procedures

45. Somatic hybridization

46. limitations
 Intergenic process between widely related plants which are not
compatible sexually are not possible
 In certain wide crosses , elimination of chromosomes from hybrid
cell is another limitation of somatic hybridization
 In protoplast fusion experiments ,the percentage of fusion
between two different parental protoplast is very low
 For hybrid identification, selection and isolation at the culture
level ,there is no standardized method which is applicable for all
materials

47. Advantages
 It facilitates the mixing of two genomes and can be used in
crosses at interspecific intraspecific or even intergenic
level
 To create new strains with desired properties and for strain
improvement
 Mixing two genomes open the door to gene transfer and a
study of gene expression , stability of several traits and cell
genetic changes

48. summary
In a nutshell, protoplast culture has found its many
uses and many applications in fields such as
genetic engineering and crop breeding. genetic
transformation by introduction of transgene DNA
somatic hybridization by protoplast fusion of
species or subspecies resistant to traditional cross
breeding and isolation of sub cellular organelles
are the examples of how the development of
protoplast system has led to an increase in the
versatility of plants.

49. Thank you

50. References
 http://www.biologydiscussion.com/protoplasts/protoplasts-importance-isolation-
culture-and-regeneration/10666
 https://en.wikipedia.org/wiki/Protoplast
 https://users.ugent.be/~pdebergh/pro/pro2_p11.htm
 http://www.biologydiscussion.com/plants/plant-protoplast/top-15-applications-of-
protoplast-culture-plant-
tissue/14747https://www.slideshare.net/HudaNazeer/protoplast-culture-53284966