Protoplast culture refers to the process in which whole plants are developed from the culture of cells without cell wall. This techniques widely used in plant breeding and crop improvement.

1. By

2. Introduction
Protoplast
 The term protoplast means spherical plasmolysed content of
plant cell covered by plasma membranes or naked cell
without cell wall
 Protoplasts are naked plant cells without the cell wall, but
they possess plasma membrane and all other cellular
components
 Protoplasts of different species can be fused to generate a
hybrid, and this process is known as protoplast fusion or
somatic hybridization Isolated
protoplast

3. Important events
• The term protoplast was first introduced by Hanstein in 1880.
• Klercker (1892) was the first to isolate protoplasts from plasmolyzed cells of Stratiotes
aloides by microsurgery on plasmolyzed cells by mechanical method.
• First documented research in protoplast was started by Cocking in 1960. He used an
enzymatic method for the removal of cell wall.
• Vasil and Vasil (1974)reported the regeneration of tobacco and Petunia plants from
protoplasts and culture of corn protoplasts.
• Hayashimoto et al. (1990) Protoplast transformation and regeneration of fertile
transgenic plants of rice (O. sativa L.) cultivars Nipponbare and Taipei 309.
• Yoo et al. (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for
transient gene expression analysis.

4. Isolation of Protoplasts
The isolated protoplast is highly fragile and the outer plasma
membrane is fully exposed. The plasma membrane is the only
barrier between the interior of the living plant cell and the
external environment.
Isolation of protoplast can be done by three methods.
(a)Mechanical (non enzymatic)
(b)Sequential enzymatic method
(c)Mixed enzymatic method (simultaneous)

5. Sources of explants for protoplast
isolation
Leaves
The leaf is the most convenient and popular source of plant
protoplast because it allows isolation of large number of relatively
uniform cell
Callus culture
Young callus culture are also ideal material for obtaining large quantities of
protoplasts because old callus cultures tend to form giant cells with cell wall
usually difficult to digest. Hence young actively growing callus is sub cultured and
used after 2week for protoplast isolation.
Suspension culture
Young cell suspensions are ideal for isolation of protoplasts in large quantities. Cell
suspension cultures may provide a very good source of protoplast.

6. Diagrammatic representation of the technique used for
isolation, culture, and regeneration of plantlets from leaf

7. Mechanical method
 Plant cells are kept in a suitable
plasmolyticum (in plasmolysed
cells, protoplasts shrink away from
cell wall) and cut with a fine knife
into small pieces.
 Then these pieces are
deplasmolysed by using dilute
solution to release protoplast.
 Generally this method is suitable
for isolation of protoplasts from
vacuolated cells of storage tissue
such as onion bulbs, scales, beet
root and radish roots.
Diagrammatic representation of the mechanical method of
protoplast isolation from epidermis
Low sucrose
solution

8. Limitation of mechanical method
 This method is not suitable for isolating protoplast
from meristematic and less vacuolated cells.
 Laborious and tedious
 Poor yield of protoplasts, and also the viability of
protoplasts is low.

9. Enzymatic method
 Protoplasts can be isolated from plant tissues or cultured cells by
enzymatic digestion to remove the cell walls.
 The enzymatic method is almost invariably used now for the isolation of
protoplasts.
 It gives large quantities of protoplasts, where cells are not broken and
osmotic shrinkage is less.
Enzymatic method of protoplast isolation can be used to types:
(a)Sequential enzymatic method
(b)Mixed enzymatic method

10. Sequential enzymatic method
 This method was first
used Takebe and others
in 1968 in two steps
 The macerated tissue
was first incubated in
pectinase (degrade
pectin cell wall) and
then treated with
cellulase (degrade
cellulosic wall) for
liberation of protoplast
Sequential
Enzymatic Method

11. Mixed enzymatic method
 This is one step procedure in which both
enzymes are used together to reduce time and
chances of contamination.
 Power and Cocking (1968) used this method for
the isolation of protoplasts from a number of
plants and it gives very good yields, as high as
2.5 × 106 protoplasts/gram of leaf tissue
 Protoplast can be isolated by treating cell, with
a suitable mixture of enzyme of cell wall
degrading enzymes. The mixture of pectinase
or macerozyme (0.1-1.9%) and cellulase (1-2%)
is suitable for majority of plant parts.

12. Commercially available enzyme for
protoplast isolation
S.No. Enzyme Source
1. Zymolyase Arthrobacter luteus
2. Cellulose onozuka Trichoderma viride
3. Rhozyme Aspergillus niger
4. Macrozyme Rhizopus arrhizus
5. Hemicellulase Aspergillus niger
6. Macerase Rhizopus arrhizus
7. Helicase Helix pomatia
8. Pectolyase Aspergillus japonica
9. Drisclease Irpex lacteus
10. Cellulysin Trichoderma viride

13. Osmoticum
Elevated osmotic concentration is require for both isolation and culture
of protoplasts. Usually by adding 500-800 mmol-1 sorbitol or mannitol to
stabilize the protoplast and to prevent them from bursting.
The osmotica are of two types-
 Non ionic osmoticum-
 The non-ionic substances most commonly used are soluble
carbohydrates such as mannitol, sorbitol, glucose, fructose, galactose
and sucrose. Mannitol, being metabolically inert, is most frequently
used.
 Ionic osmoticum-
 Potassium chloride, calcium chloride and magnesium phosphate are
the ionic substances in use to maintain osmotic pressure.
 Usually 50-100mM l-1 CaCl2 is added to the osmoticum as it improves
plasma membrane stability.

14. Viability testing of isolated protoplast
 The isolated protoplast should be healthy
and viable in order to undergo proper
division and regeneration.
 This can be done by microscopic
observation of untreated cell or staining
the cells with suitable chemicals to
indicate active metabolism in the
protoplasts.

15. Phase contrast microscopy
 Cytoplasmic streaming movement (cyclosis) and
the presence of clear, healthy nucleus indicate
the cells are in viable state.
 For this phase contrast microscope is better
because observation of unstained cells under
bright field is highly difficult

16. Tetrazolium test
 Respiratory efficiency of cell is measured by reduction
of 2,3,5- triphenyl tetrazolium chloride (TTC) to the
red dye formazan.
 The formazan formed can be extracted and measure
spectrophotometrically

17. Fluorescein Diacetate Method
 The 0.5% fluorescein diacetate (FDA) in acetone is
prepared and stored at 40C. This was added at 0.01% of
final concentration to protoplasts suspension with
osmotic stabilizer.
 After 5 minutes incubation the cells are observed under
microscope with suitable filter.
 Fluorescence microscopy detects the dye accumulates
inside viable protoplasts

18. Evans blue staining
 The 0.025% of Evans blue stain solution was
used for staining the protoplasts. The stain
gives color to dead protoplasts by
becoming permeable to dead ones.
 Viable protoplasts remains colourless due
to impermeability of plasma membrane to
the stain.

19. Culture medium
 Generally MS and B5 media with suitable modifications are used.
 The medium for protoplast culture should be devoid of ammonium, and the quantities of
iron and zinc should be less.
 The concentration of calcium should be 2–4 times higher used for cell cultures. This is
needed for membrane stability.
 The vitamins used for protoplast cultures are the same as used in standard tissue culture
media.
 Glucose is the preferred carbon source by protoplasts although a combination of sugars,
such as glucose and sucrose, can be used.
 High auxin/kinetin ratio is suitable to induce cell divisions, while high kinetin/auxin ratio is
required for regeneration.

20. Culture of Protoplast
 First step in the protoplast culture is the
development of a cell wall around the
membrane of isolated protoplast. This is
followed by induction of divisions in the
protoplast-derived new cell giving rise to
small cell colony.
 Cell wall formation can be detected by
staining with 0.1% calcofluor white(CPW)
fluorescent stain. Calcofluor white (CFW)
stain binds to the newly formed cell walls
which emit fluorescence.
 Isolated protoplasts of their hybrid cells
are cultured either in a liquid or agar
medium.
 The culture dish is then maintained at low
light or dark conditions at 25-280C
Fig. Plant regeneration from protoplasts of Gentiana straminea
Maxim. using agar-pool culture. Freshly isolated protoplasts
from embryogenic calli (a); Viability of freshly isolated
protoplasts stained by FDA (b); First cell division of protoplast
(c); Second cell division of protoplasts (d); Third cell division of
protoplasts (e); Microcolony formation (f); Microcalli formation
(g); Embryogenic callus formation from protoplast-derived cell
colony (h); Somatic embryos at different development stages
(i-k); Regenerated plantlet (l). (a-f: bar = 50 μm; g: bar = 1
cm; h-l: bar = 1 mm). (Source – G. Shi et al. 2016)

21. Protoplast culture and regeneration
 As the cell wall formation around protoplasts is
complete, the cells increase in size.
 The protoplasts, which are capable of dividing,
undergo first division within 2–7 days and form
small cell colonies after 2–3 weeks.
 With suitable manipulations of nutritional and
physiological conditions, the cell colonies may be
grown continuously and form visible colonies
(macroscopic colonies).
 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 embryogenic
differentiation to finally form the whole plant. Major steps of protoplast isolation, culture and
regeneration of plant

22. Protoplast fusion and Somatic hybridization
 Protoplast fusion facilitates mixing of two genomes
and could be exploited in crosses which are not
possible by conventional techniques due to
incompatibility
 Protoplast fusion can be used to make crosses
within species (intra specific), between species inter
specific , within genera (intrageneric) and between
genera (intergeneric)
Protoplast fusion may be of 3 kinds.
1.Spontaneous fusion
2.Mechanical fusion
3.Induced fusion

23. Spontaneous fusion
 In spontaneous fusion, the adjacent protoplast
in enzyme mixture have tendency to fuse
together to form homokaryons (having same
type of nucleus)

24. Mechanical fusion
 Gentle tapping of protoplasts suspension in a
depression slide result in protoplast fusion.
 The giant protoplasts of Acetabularia have been
fused mechanically by pushing together two
protoplasts. This fusion does not depend upon the
fusion inducing agent

25. Induced fusion
 Freshly isolated protoplast can be induced to
undergo fusion with help of a range of fusogens
eg. NaNO3, high ph/Ca++, PEG, electrofusion.
 The following treatment have yielded success in
producing somatic hybrid plants.

26. NaNO3 treatment
 Induced fusion by NaNO3 was first demonstrated by
Power et al. 1970. Isolated protoplasts were
cleaned by floating in sucrose osmoticum.
 Transfer of the protoplast in 0.25M NaNO3 solution
and subsequent centrifugation promoted the fusion
process.
 This procedure result in a low frequency of
heterokaryon formation and protoplasts are
markedly altered in their uptake capabilities.

27. High pH/Ca++ Treatment
 This method was developed by Keller and Melchers
(1973) for fusing two different lines of tobacco
protoplast.
 Isolated protoplasts are incubated in a solution of
0.4 M mannitol containing, 0.05M CaCl2, with pH at
10.5 (0.05M glycine- NaOH buffer) and temperature
at 370C.
 Aggregation of protoplasts generally take place at
once and fusion occurs within 10 min.
 Many intraspecific and interspecific somatic hybrids
have been produced using this procedure.

28. Polyethylene glycol (PEG) induced protoplast fusion
 PEG has been used as a fusogen in a number of plant
species because of the reproducible high frequency
of hetrokaryon formation.
 About 0.6 ml (28-50% PEG)(MW 1500-6000) PEG
solution is added in the protoplast mixture in the
tube.
 After having capped the tube, protoplast in PEG are
incubated at room temperature for 30-40 minutes.
 Gradual washing of the protoplast to remove PEG by
the addition of 0.5 -1ml of protoplast culture
medium in the tube after every 10 min.
 The washing medium may be alkaline (pH- 9-10) and
contain a high Ca+2 ion concentration (50m mol-1).
This approach is a combination of PEG and high pH/
Ca+2 treatments, and is usually more effective than
either treatment alone.
 PEG either provides a bridge by which Ca+2 can bind
membrane surface together or leads to a disturbance
in the surface charge during the elution.
PEG
dilution
+
PEG
28-50%
PEG
dilution
Heterokaryon
Hybrid cell
PEG induced Protoplast Fusion

29. Electrofusion techniques
 The electrofusion technique, which utilizes low voltage
electric current pulses to align the protoplast in a single row
like a pearl chain.
 The aligned protoplasts are pushed, with a
micromanipulator, and pairs of protoplast may be isolated in
individual microelectrofusion chambers.
 The pairs of protoplast can be fused by a very brief (few
microsecond ) pulse of high voltage (500-1000 Vcm-1).
 The high voltage creates transient disturbance in the
organization of plasmalemma, which leads to the fusion of
neighbouring protoplast.
 The entire operation is carried out manually in a specially
designed equipment called electroporator.

30. Selection of Hybrid cells
 During the process of protoplast fusion there is a
possibility of formation of Homokaryons, Heterokaryons,
and unfused parental protoplasts are present in the
mixture.
 Hence proper selection of Hybrid protoplasts or cell is of
most important.
 In somatic hybridization experiment only hybrid protoplast
particularly those resulting from fusion between one
protoplast of each of two species are of interest.
 Different method can be used to select fusion products of
protoplasts that have distinct physical characteristics.

31. Drug sensitivity and Resistance
 In Petunia, there are two species namely, P. hybrida
protoplast forms macroscopic callus on MS medium and
sensitive to antibiotic actinomyocin –D. Where P. Paroddii
forms cell colonies and resistant to actinomyocin D.
 The fusion protoplasts of these two species will be having
character of both i.e they form macroscopic colonies and
resistant to actinomyocin-D on the MS supplemented with
actinomyocin–D thus helps in selection.
 But parental protoplast form either smaller colonies (P.
Paroddii ) or fails to divide and form the colonies (P. hybrida)
because of inhibitory effect of antibiotic.

32. Auxotrophic mutant
 The Original protoplasts have the capacity to grow
in minimal medium is known as prototroph.
 The mutants of Prototrophs which is not having the
capacity to grow in the minimal media is known as
Auxotroph.
 The hybrid protoplast are known to grow in
minimal medium and parental protoplast are not
able to grow in minimal medium. It helps in
selection process.

33. Visual selection
• In this selection method the fused protoplast are identified by
fusing the chlorophyll rich parent with chlorophyll deficient
parent.
• The products of fusion are identified by using microscope
because hetrokaryons are bigger and green in colour, whereas
parental protoplasts are either small and colourless.
• This is further differentiated by using suitable selective
medium which support good growth of only hybrid cell.

34. Fluorescent Labels
 In this method fluorescent labeled dyes are used to detect the
fusion product.
 If the two original protoplast cultures are pre incubated for
12-15hr, one in octadecanoyl aminoflurescein and the other in
octadecyl palamine B each group of protoplasts takes on a
specific fluorescence colour. The dye are non toxic and do not
affect viability , wall regeneration or growth.
 After fusion of the protoplasts fusion products may be
identified their fluorescence characteristics under a
fluorescence microscope.

35. Cybrids
Cybrids are cells or plants containing nucleus of
one species but cytoplasm from both the parental
species.
Cybrid formation
(a) Fusion of a normal protoplast of one species
with an enucleate protoplast or a protoplast
having an inactivated nucleus of the other
species.
(b) Elimination of the nucleus of one species from
a normal heterokaryon.
(c) Gradual elimination of the chromosomes of
one species from a hybrid cell during the
subsequent mitotic divisions.
(d) The cybrid approach used for cytoplasmic male
sterility from N. tabacum to N. sylvestris,
from Petunia hybrida to P. axilaris.

36. Application
 To overcome sexual incompatibility
Production of novel interspecific and intergeneric crosses between plants that
are difficult or impossible to hybridize conventionally. Therefore, sexual
incompatibility may be overcome.
 Cybrid Production
 Transfer of plasmagenes of one species into the nuclear background of
another species in a single generation .
 Sexually incompatible combinations.
 Recovery of recombinants between the parental mitochondrial or
chloroplast DNAs.
 Uptake of foreign materials
Being naked in nature, isolated protoplasts are considered to be very suitable
for genetic and cytoplasmic modifications. It has been reported that plant
protoplast has a unique property in uptaking isolated nuclei, DNA,
chromosomes, chloroplasts, cyanobacteria, nitrogen fixing bacteria, and virus
particles.

37. Genus Parent species and their chromosome
number
Chromoso
me number
of hybrid
Brassica B. oleracea (2n = 18) + B. campestris (2n = 20)
B. napus (2n = 38) + B. oleracea (2n = 18)
B. napus (2n = 38) + B. nigra (2n = 16)
B. napus (2n = 38) + B. carinata (2n = 34)
Wide
variation
Nicotiana N. tabacum (2n = 48) + N. alata (2n = 18)
N. tabacum (2n = 48) + N. glauca (2n = 14)
N. tabacum (2n = 48) + N. rustica (2n = 48)
N. tabacum (2n = 48) + N. octophora (2n = 24)
N. tabacum (2n = 48) + N. mesophila (2n = 48)
N. tabacum (2n = 48) + N. glutinosa (2n = 24)
66-71
72
60-91
48
96
50-88
Table . Examples of Somatic Hybridization of Interspecific hybrids
in different Plants

38. Genus Parent species Somatic hybrid
family
Raphanus × Brassica
Raphanus sativus + B.
oleracea
Raphanobrassica
Moricandia ×
Brassica
Moricandia arvensis +
B. oleracea
Moricandiobrassica
Eruca × Brassica
Eruca sativa + B.
napus
Erucobrassica
Nicotiana ×
Lycopersicon
Nicotiana × Petunia
Nicotiana tabacum +
Lycopersicon
esculentum
Nicotiana tabacum +
Petunia inflorata
Nicotiopersicon
Nicotiopetunia
Solanum ×
Lycopersicon
Solanum tuberosum +
Lycopersicon
esculentum
Solanopersicon
Datura × Atropa
Datura inoxia +
Atropa belladonna
Daturot
Table Examples of Somatic Hybridization of Intergeneric
hybrids in different Plants