All about what is protoplast, how to isolate protoplast & technques to culture them. Also the potent applications & somatic hybridization.

1. 1
DEPARTMENT OF BIOTECHNOLOGY
Presented By ZAINAB SHAHID
2ND YEAR STUDENT
SUBMITTED TO: Mr. PRAMOD KUMAR
(HOD BIOTECHNOLOGY )

2. 1. Introduction to Plant cell
2. Plant protoplasts
3. Isolation of protoplasts
4. Mechanical isolation
5. Enzymatic isolation
6. Purification of isolated protoplasts
7. Protoplasts viability and density
8. Protoplast culture
9. Application of protoplasts
10. Somatic hybridization.
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3. 5

4. It consists of three types of layers;
The middle lamella :
Pectic compounds and proteins.
The primary wall:
Cellulose and hemicelluloses.
The secondary wall:
Cellulose, hemicelluloses and lignin.
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5. http://www.google.com.pk/search?q=cell+wall+plant&source=lnms&tbm=isch&sa=X
&ei=h9GxUYa3BcyuPM7wgfgP&ved=0CAcQ_AUoAQ&biw=1366&bih=624#imgrc=_
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6.  Large molecules cannot pass through
the cell wall.
 Somatic hybridization and genetic
manipulation cannot be done.
,So we have to remove the cell wall.
After removing it we get Protoplasts
and Spheroplasts.
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7. PROTOPLAST…
 Unit of biology which is composed of
a cell's nucleus and the surrounding
protoplasmic materials. Hanstein
(1880).
 It is a plant cell that has its cell wall
completely removed using either
mechanical or
enzymatic means. 7

8. 8http://www.biotechnologyforums.com/thread-1853.html

9. PlantTissue Cells Plasmolysis
Microscope Observation of cells
Cutting cell wall with knife Release of protoplasts
Collection of protoplasts
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10.  Isolation of Protoplast
 (Separation of protoplasts from plant tissue)
1. Mechanical Method 2. Enzymatic Method
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11. 11http://www.google.com.pk/search?q=plant+cell&source=lnms&tbm

12. • Used for vacuolated cells like onion
bulb scale, radish and beet root
tissues.
• Low yield.
• Laborious and tedious process.
• Low viability.
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13. • Cocking 1960.
• Used for variety of tissues and organs
including leaves, petioles, fruits, roots,
coleoptiles, hypocotyls, stem, shoot apices,
embryo microspores.
• Osmotic shrinkage is minimum.
• Cells remain intact and are not injured.
• Protoplasts are readily obtained.
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14.  Leaf sterilization,
 Removal of epidermis
Plasmolysed
cells
Plasmolysed
cells
Protoplast released Release of
isolated cells
Isolated
Protoplast
Pectinase +cellulase
Pectinase
Protoplast
released
cellulase
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15. 1. Incubation of leaf segments over night,
2. Treated with enzymes to liberate
protoplasts, Mixture is filtered,
3. Centrifugation,
4. Protoplast forms pellet,
5. These are washed with sorbitol 3X,
Centrifugation,
6. Cleaned protoplast float,
7. Pipettes out. 15

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17. 1. Two enzyme mixtures (mixture A and 'mixture
B) are used one after the other.
2. Leaf segments with mixture A (macerozyme in
manifold at pH 5.8) are vacuumed infiltrated for
5 min., transferred to a water bath at 25°C and
subjected to slow shaking.
3. After 15 min. the enzyme mixture is replaced by
fresh 'enzyme mixture A' and leaf segments are
incubated for another hour.
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18. 4. The mixture is filtered using nylon mesh,
centrifuged for 1 min.
5. Washed three times with 13% mannitol to get a
pure sample of isolated cells.
6. Cells are then, incubated with 'enzyme mixture
B' (cellulase in a solution of mannitol at pH 5.4)
for above 90 min, at 300 C.
7. After incubation, the mixture is centrifuged for
1 min, so that protoplasts form a pellet, which
are cleaned three times as in 'one step method.
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19. • Protoplasts are separated after digestion period from
enzymes and cellular debris, transfer to the suitable
medium.
• A concentrated solution of mannitol, sorbitol and
sucrose
• (0.3-0.6M) can be used as a gradient.
• This is done by centrifugation at low speed followed by
filtration through nylon mesh (60-70µm).
• Again followed by centrifugation or by density gradient
centrifugation step.
• This pallet is dissolved.
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20. 20http://www.google.com.pk/search?q=plant+cell&source

21. To check the viability of isolated protoplasts, viability tests are used. Like
1. Fluorescein diacetate:
It accumulates only inside the plasma lemma of viable protoplasts,
can be detected with fluorescence/UV microscopy.
2 . Evans blue:
Intact viable protoplasts, exclude the Evans blue stain.
Impermeability of the cell to Evans blue indicates a living cell.
3. Cyclosis: or protoplasmic streaming can be a measure of
viability.
 The density of the protoplasts in the suspension (number/unit
volume) is determined by counting with a modified haemocytometer
such as Neubauer with a field depth of 0.2mm.
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22. 22http://www.google.com.pk/search?q=plant+cell&source

23. Isolated protoplasts can be cultured in an
appropriate medium to reform cell wall and
generate callus.
Optimal culture conditions:
1. Optimal density to the culture.
at low density protoplasts will lose soluble cell components.
2. Optimal auxin to cytokynin ratio, glucose and sucrose.
3. Maintain the osmoprotectant in the medium until the cell wall
has reformed.
4. 20-28 °C, pH5.5-5.9, 0.25% casein hydrolysate, BAP and NAA.
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24. 1. Hanging drop culture.
2. In the wells of microtitre plates.
3. On the semisolid medium using
agarose plates.
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25. Hanging drop culture
Culturing protoplasts in
droplets(100 micro litter),
Suspended on the lid of a
Petri dish, with sterile water
in the base to provide
humidity.
Because small volumes are
required , this arrangement
is a convenient way of
establishing optimal
conditions of growth.
25
http://www.google.com.pk/search?q=plant+cell&sour
ce

26. 26http://www.google.com.pk/search?q=plant+cell&source

27. On a semi-solid medium, agarose plate
27http://www.google.com.pk/search?q=plant+cell&source

28.  On a semi- solid medium using agarose plates is
an effective method as it provides a supporting
matrix for the protoplasts.
 Standard agar is toxic to protoplasts and low
temperature gelling agarose is used instead.
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29.  It provides growth factors that stimulates wall
synthesis and cell division.
 The formation of cell wall can be followed by
using the compound 0.1% Calcofluor White.
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30. 30http://www.google.com.pk/search?q=plant+cell&source
The formation of cell wall can be followed by using the compound o.1% Calcofluor
White.

31. Protoplasts can be used to study;
 Metabolic studies including photosynthesis.
 For DNA transformation.
 For somatic hybridization.
 Ingesting "foreign" material into the cytoplasm.
 Single cell systems.
 Wall synthesis and deposition.
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32. 32
Fusion of protoplasts
Ingesting of "foreign" material
wall synthesis and deposition single cell systems

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34.  Fusion of protoplasts facilitates mixing of
two whole genomes and could be exploited
in crosses at interspecific, intergeneric, or
even interkingdom levels.
 Isolated protoplasts are devoid of walls
make them easy tools for undergoing
fusion in vitro.
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35. Somatic hybridization technique
1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants
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36. 36
http://onlinelibrary.wiley.com/doi/10.1002/9780470650189.ch6/summary

37. Protoplast Fusion
(Fusion of protoplasts of two different genomes)
Spontaneous Fusion Induced Fusion
Intraspecific Intergeneric Chemo fusion Mechanical
Fusion
Electro fusion
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38.  Protoplasts fuse spontaneously during
isolation process mainly due to physical
contact.
Intraspecific produce homokaryones.
Intergeneric have no importance.
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39.  Chemo fusion- fusion induced by chemicals
• Types of fusogens
1. PEG
2. NaNo3
3. Ca 2+ ions
4. Polyvinyl alcohol
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40. The fusion process
Electro fusion: Protoplasts are aligned in a
special chamber, electric current is
applied(100kV/m), opening channels in cell
membrane.
PEG fusion: (Polyethylene glycol);
 Protoplasts are coated with PEG, then
incubated together; where cell membranes fuse,
channels begin to form
After fusion, "fusion products" begin to "round
up"
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41. 1. Production of novel interspecific and
intergeneric hybrids.
Pomato (Hybrid of potato and tomato).
2. Production of fertile diploids and polyploidy
from sexually sterile haploids and triploids.
3. Transfer gene for disease resistance, a biotic
stress resistance, herbicide resistance and
many other quality characters.
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42. Advantages of somatic hybridization:
• Production of heterozygous lines in the
single species which cannot be propagated
by vegetative means.
• Formation of cytoplasm containing
organelles of two different parents.
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43. 1. Poor regeneration of hybrid plants.
2. Non-viability of fused products.
3. Not successful in all plants.
4. Production of unfavorable hybrids.
5. Lack of an efficient method for selection of
hybrids.
6. No confirmation of expression of particular
trait in somatic hybrids. 43
zainab.agra@gmail.com

44. Advantages of somatic hybridization:
• Production of heterozygous lines in the
single species which cannot be propagated
by vegetative means.
• Formation of cytoplasm containing
organelles of two different parents.
44

45. Advantages of somatic hybridization:
• Production of heterozygous lines in the
single species which cannot be propagated
by vegetative means.
• Formation of cytoplasm containing
organelles of two different parents.
45

46. 1. Poor regeneration of hybrid plants.
2. Non-viability of fused products.
3. Not successful in all plants.
4. Production of unfavorable hybrids.
5. Lack of an efficient method for selection of
hybrids.
6. No confirmation of expression of particular
trait in somatic hybrids.
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