protoplast culture ,

1. SOMATIC HYBRIDIZATION

2. Protoplast Culture: definition
Isolated protoplasts have been described as "naked"
cells because the cell wall has been removed by either a
mechanical or an enzymatic process. In the isolated
protoplast the outer plasma membrane is fully exposed

3. Development of hybrid plants through
the fusion of somatic protoplasts of two
different plant species/varieties is called
somatic hybridization

4. 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

5. Isolation of protoplast from leaves
 Leaves are most commonly used
Step are
1) Sterilization of leaves
2) Removal of epidermal cell layer
3) Treatment with enzymes
4) Isolation of protoplast
Purification of protoplast
Viability of protoplast

6. Isolation of Protoplast
(Separartion of protoplasts from plant tissue)
1. Mechanical Method 2. Enzymatic Method

7. 1. Mechanical Method
Plant Tissue
Collection of protoplasm
Cells Plasmolysis
Microscope Observation of cells
Cutting cell wall with knife Release of protoplasm
Klercker 1892 (cited in Cocking 1972)

8.  Suppose an animal or a plant cell is placed in a solution of sugar
or salt in water.
 If the medium is hypotonic — a dilute solution, with a higher water
concentration than the cell — the cell will gain water through
osmosis.
 If the medium is isotonic — a solution with exactly the same water
concentration as the cell — there will be no net movement of
water across the cell membrane.
 If the medium is hypertonic — a concentrated solution, with a
lower water concentration than the cell — the cell will lose water
by osmosis.
 Plasmolysis is the process in plant cells where
the cytoplasm pulls away from the cell wall due to
the loss of water through osmosis. This occurs in
ahypertonic solution

9. 1. Mechanical Method
 Used for vacuolated cells like onion bulb
scale, radish and beet root tissues
 Low yield of protoplast
 Laborious and tedious process
 Low protoplast viability
 Restricted to tissues having large
vacuolated cells.

10. 2. Enzymatic Method
Leaf sterlization, removal of
epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase Pectinase
Protoplasm released
Release of
isolated cells
cellulase
Protoplasm
released
Isolated
Protoplasm
(Cocking 1960) Isolation of protoplast from
root tips of tomato
(Lycopersicon esculentum)

11. Enzymes for protoplast isolation
The enzymes that can digest the cell walls are required for protoplast isolation.
Chemically plant cell – composed- cellulose, hemicellulose,, pectin
So req- enzymes- cellulase, hemicellulose, pectinase
In fact, the various enzymes for protoplast isolation are commercially available.
The enzymes are usually used at pH 4.5 to 6.0, temperature 25-300C with a wide
variation in incubation temp and that may range from half an hour to 20 hrs
The enzymatic approach by two ways:
a)Two step or sequential method- pectinase (to separate cells by degrading
middle lamella and then cellulase removes the cell wall proper)
b) One step or simultaneous method- Simultaneously both macroenzyme and
cellulase

12. •Obtain sterile plant
material
•Rinsing in a suitable
osmoticum
•Facilitating enzyme
penetration
•Sequential or
spontaneous
•Purification of the
isolated protoplasts
(removal of enzymes
and cellular debris)
•Transfer to a
suitable medium
Use of enzymes results in a high yield of uniform protoplasts after
removal of cellular debris Protoplasts can originate from different sources:
greenhouse or field material, micropropagated plants, calli,

13. •Healthy leaves, removed from the plant and washed, sterilized and rewashed in sterile
distilled water (subsequent procedures are conducted under aseptic conditions).
When leaves are in the final rinse, lower epidermis is peeled from the leaves or the lower
epidermis is scored several times.
Cut the leaves into small sections, and transfer to filter sterilized enzyme solution.
Seal the dishes wrap them with aluminum foil (leave overnight).
Teased gently with forceps to release the protoplasts.
Purify protoplasts (filtration, centrifugation, and washing)

14. Enzymatic Method
 Used for variety of tissues and organs
including leaves, petioles, fruits, roots,
coleoptiles, hypocotyls, stem, shoot apices,
embryo microspores
 Mesophyll tissue - most suitable source
 High yield of protoplast
 Easy to perform
 More protoplast viability

15. •Protoplasts are filtered through a nylon mesh
(64micrometer) to remove undigested tissue,
cell clumps, and cell wall debris.
•Transfer filtrate to centrifuge tube and spin at
+ 75 x g (5 min).
•Debris (in supernatant) is carefully removed
(protoplasts have formed a pellet at the base
of the tube).
•Protoplasts are carefully resuspended in
culture medium (plus 13% mannitol), and the
process is repeated three times.
•Protoplasts are examined for density and
viability.
Protoplast purification

16. •Fluorescein diacetate:
accumulates only inside
the plasmalemma of viable
protoplasts, can be detected
with fluorescence/UV
microscopy
•Phenosafranine stainig: used at
0.01% conc and specific for dead
protoplast that turn red. Viable
protoplast remain unstained
•Evans blue: Intact viable
protoplasts, exclude the
Evans blue stain.
Impermeability of the cell
to Evans blue indicates
a living cell.
Checking of viability of protoplast

17. Protoplast Culture
The very first step in protoplast culture is development of a cell wall
around the membrane of the protoplast.
Protoplasts can been cultured in several ways:
•Hanging-drop cultures
•Microculture chambers
•Soft agar (0.75 % w/v) matrix.
This is one of the better methods
as it ensures support for the protoplast.

18. Culture media
A) Nutritional components :
 medium should be devoid of ammonium and quantities of iron and
zinc should be less,
 The concentration of calcium should be 2-4 times higher than used for
cell culture. This is needed for membrane stability.
 High auxin/kinetin ratio is suitable to induce cell division while high
kinetin/auxin ratio is required for regeneration.
 Carbon source is normal as used and vitamins used for protoplast
cultures are the same used in standard tissue culture media.

19. B) Osmoticum:
Protoplast released directly into standard cell culture medium will burst
Broadly refers to the reagents/chemicals that are added to increase the
osmotic pressure of a liquid.
Isolation and culture of protoplast require osmotic protection until they
develop a strong cell wall.
If freshly isolated protoplasts are directly added to culture medium they
will burst.
Osmoticum: 0.3 to 0.7 M of sugar solution., mannitol or sorbitol.

20. Culture methods
 Liquid culture
 Liquid Droplet method
 Hanging Droplet method
 Feeder layer
 Co-culturing

21. Regeneration of protoplast

22. SWEET ORANGE SUSPENSION CULTURE PROTOPLASTS

23. RMAN

25. From an idea to generation

26. Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous Fusion 2. Induced Fusion
Intraspecific
(Callus)
Intergeneric Electrofusion
Mechanical
Fusion
Chemofusion

27. 1.Spontaneous Fusion
 Protoplast fuse spontaneously during isolation
process mainly due to physical contact
• Intraspecific produce homokaryones
• Intergeneric have no importance

28. 2. Induced Fusion
 A) Chemofusion - fusion induced by chemicals
• Types of fusogens
• PEG
• NaNo3
• Ca 2+ ions
• Polyvinyl alcohol
• Lysozyme
• Dextran
• Fatty acids and esters.

29.  Of these , the following treatments have yielded success in producing somatic hybrid
plants.
 1 NaNOӡ treatment : first demonstrated by power et al.(1970)
 Isolated protoplasts cleaned by floating in 10% sucrose solution .
 Transfer of protoplast
 in 5.5% NaNOӡ solution
 incubation then centrifugation @2000rpm
protoplast kept in water bath for 300C for 30 min during which fusion occurs
 This treatment results in low frequency of heterokaryon formation ,
particularly when mesophyll protoplast are used.


30.  2. High pH and high Ca++ treatment : Method was developed
by Kelier and Melchers(1973) for fusing two different lines of tobacco
protoplast and now commonly used.
 Isolated protoplast
 incubated in
 solution of 0.5M mannitol containing 0.05M CaCl2,
 pH 10.5 and temp. 37ºC
 (30-40 min.)

 aggregation of protoplast and fusion usually occurs
 with in 10 min.
 By this methods , 20-50% of the protoplasts involved in fusion.

31.  3. PEG treatment : This has the become the method of choice, due to its
high success rate. Kao and Michayluk (1974).
 Isolated protoplasts in culture medium (1ml) are mixed with equal volume (1ml) of
28-56% PEG in a tube.
 PEG enhances fusion of protoplasts in several species.
 tube is shaken(5 min) and allowed to settle(10 min).
 settled protoplasts washed several times with culture medium.
 This method is widely used for protoplast fusion.
 Advantage : a) high frequency of heterokaryon formation
 b) low toxicity to cells.
 c) reduced formation of binucleate heterokaryon

32. Mechanism of fusion
Protoplast fusion: PEG
a) Agglutination (Adhesion)

33. Schematic representation of 3 most successful protoplast fusion
strategies.
 Protoplast of species(A) + Protoplast of species(B)
( suspended in enzyme mixture)
PEG induced fusion Electrofusipn
High Ca 2+
High pH treatment PEG(28-58%) (high MW)(inc. agg.) Low voltage
15-30 min.
Protoplast aggregation Protoplast
Ca 2+ 50mM/1
chain formed , centrifuge- 3min, 500 rpm
pH 10.5, temp.37˚C.
Washing medium High Voltage i
40-50 min incubation pH 9-10 ( few micro second)
Ca 2+ 50mM/1
water bath
Protoplast fusion

35. induced fusion contd….
 B) Mechanical Fusion- Physical fusion of protoplasts under
microscope by using micromanipulator and perfusion micropipette.
 C) Electrofusion- Fusion induced by electrical stimulation.
• Pearl chain of protoplasts is formed by low strength electric field
(10kv m-1)
• Fusion of protoplasts of pearl chain is induced by the application of
high strength electric field (100kv m-1) for few microsecond.
• So the plasma lemma distribution and organization disturbances
leading to the fusion of two protoplasts and the fusion of these
protoplast is done in device electroporator.

36. Protoplast fusion: Electrofusion
Low intensity AC current to
protoplast suspension
750-1000 V/cm for short
duration 20-50 µsec

37. 2.2 Protoplast fusion: Electroporation

38. Selection of hybrid cells
 Protoplast suspension recovered after a treatment with a fusion inducing
agent (fusogen) consists of following cell types : -
 1) Unfused protoplast of two species / strain.
 2) Products of fusion between two or more protoplasts of the same
species(homokaryon).
3) Hybrid protoplasts produced by fusion between one (or more) protoplast
(s) of each of the two species.(heterokaryon).
Therefore , a specific strategies has to be employed for their identificaton and
isolation. This step is called Selection of hybrid cells.

39. Identification and Selection of
somatic hybrid cells
 Hybrid identification- Based on difference between
the parental cells and hybrid cell with respect to
• Pigmentation
• Cytoplasmic markers
• Fluorochromes like FITC (fluoroscein isothiocyanate)
and RITC (Rhodamine isothiocyanate) are used for
labelling of hybrid cells(0.5 mg/l prior incubation time)
• Presence of chloroplast
• Nuclear staining
• Heterokaryon is stained by carbol-fuschin, aceto-
carmine or aceto-orcein stain

40. Hybrid Selection
(Several markers are used )
• Genetic complementation
• Phytotoxins
• Specific amino acid
• Auxin autotrophy
• Antibiotics
• Auxotrophic and metabolic mutants
• Chromosomal analysis
• Herbicides

41. Culture of the hybrid cells
Hybrid cells are cultured on suitable medium
provided with the appropriate culture
conditions.

42. Regeneration of hybrid
plants
 Plants are induced to regenerate from hybrid calli .
 These hybrid plants must be at least partially fertile, in
addition to having some useful property, to be of any
use in breeding schemes.

43. Somatic hybrid- two types :
 1) Symmetric hybrids : Some somatic hybrid plants retain the full
somatic complements of the two parental species; these are called
Symmetric hybrids .
 2) Asymmetric hybrids: Many somatic hybrids exhibit the full
somatic complement of one parental species while all or nearly all
chromosomes of the other parental species are lost during the
preceding mitotic divisions, such hybrid are referred as Asymmetric
hybrids.

44. Advantages of somatic
hybridization
 Production of novel interspecific and intergenic hybrid
 Pomato (Hybrid of potato and tomato)
 Production of fertile diploids and polypoids from
sexually sterile haploids, triploids and aneuploids
 Transfer gene for disease resistance, abiotic stress
resistance, herbicide resistance and many other quality
characters

45. Advantages of somatic
hybridization
 Production of heterozygous lines in the single species
which cannot be propagated by vegetative means
 Studies on the fate of plasma genes
 Production of unique hybrids of nucleus and cytoplasm

46. Limitations of Somatic
hybridization
 Poor regeneration of hybrid plants
 Non-viability of fused products
 Not successful in all plants.
 Production of unfavorable hybrids
 Lack of an efficient method for selection of hybrids
 No confirmation of expression of particular trait in somatic
hybrids

47. NEW SOMATIC HYBRID PLANT

48. Cybrids
 Cybrids are cells or plants containing nucleus of
one species but cytoplasm from both the
parental species .

50. Cybrid of “Murcott” (the Honey tangerine) containing
the mtDNA CMS of G1 Satsuma mandarin

51. The End
Thank you for your
Attention
51

52. Easy to start
Challenging to grow
Start HereKeep in mind the
“Indian context”
Understanding regulation is critical
Building the right team is critical

53. Power of idea is
great but it’s all about
execution
Most obvious is the
most hidden
Don’t get married to your ideas
Find what sells over and over again
Don’t presume
Get feedback early on

54. 54
Er. Shivam Sharma
shivam.shoolini@gmail.com