Download cell suspension and pathogen elimination for class

A. Suspension culture is a type of culture in which single cells or
small aggregates of cells multiply while suspended in agitated
liquid medium. It is also referred to as cell culture or cell
suspension culture
B. History W.H Muir (1953): reported fragment of Tagetes erecta
and Nicotiana tabaccum could be cultured as cell suspensions
L.Nickel (1956) described the continuous growth of a variety
of Phaselus Vulgaris
F.C.Steward and E.M. Stanz (1956): reported suspension
cultures from carrot root explant and obtained very large number
of plantlets from the culture
A. Callus grows as a mass--- difficult --- to study
cellular
events
during
its
growth
development--- chemically defined liquid
media will free cells can study
A. Cell-suspension cultures
• When friable callus is placed into the appropriate liquid medium and
agitated, single cells and/or small clumps of cells are released into the
medium and continue to grow and divide, producing a cell-suspension
culture. This callus breaks up and readily disperses
• The inoculum used to initiate cell suspension culture should neither be
too small to affect cells numbers nor too large too allow the build up of
toxic products or stressed cells to lethal levels.
Separation of cells following cell division.
Good suspension :
Culture consisting of a high percentage of single cells and small
clusters of cells.
Different requirements for different cases.
The choice of Suitable conditions is largely determined by trial
and error.
Cell suspension culture
• When callus pieces are agitated in a liquid
medium, they tend to break up.
• Suspensions are much easier to bulk up
than callus since there is no manual
transfer or solid support.
• Large scale (50,000l) commercial
fermentations for Shikonin and Berberine.
Characteristics of plant cells
• Large (10-100M
long)
• Tend to occur in
aggregates
• Shear-sensitive
• Slow growing
• Easily contaminated
• Low oxygen demand
(kla of 5-20)
• Will not tolerate
anaerobic conditions
• Can grow to high cell
densities (>300g/l
fresh weight).
• Can form very
viscous solutions
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Different time periods for subculturing
Some plants max. cell density is reached within
about 18-25 days.
In some plants as short as 6-9 days
Nylon net or stainless steel filter is used to
remove larger cell aggregates.
Small portion is withdrawn and cell density will
be checked
9-15×103 cells / cm3 for sycamore.
The best speed for 100-120 rpm
Liquid should be filled 20% of the size of the
flask for adequate aeration.
Introduction of callus into suspension
• ‘Friable’ callus goes
easily into suspension
–
–
–
–
2,4-D
low cytokinin
semi-solid medium
enzymic digestion with
pectinase
– blending
• Removal of large cell
aggregates by sieving
• Plating of single cells
and small cell
aggregates - only
viable cells will grow
and can be reintroduced into
suspension
Originates with a random critical event when plant
cells are exposed to suspension culture early on:
cells undergoing transition will have the
following characteristics:
1. A high degree of cell separation
2. Homogenous cell morphology
3. Distinct nuclei and dense cytoplasm
4. Starch granules
5. Relatively few tracheary elements
6. Doubling time 24-72 hours
7. Loss of totipotency
8. Hormone habituation
9. Increased ploidy levels
Cell suspension
culture
Big pieces eliminated and after 2-3 weeks
only singe cells or cell aggregates
transferred to new media.
Suspension can be propagated by subculturing
Ideally single cell cultures are desirable but
hard to achieve. But can be achieved if the
process of cell division, enlargement and
differentiation is synchronized
Cell suspension
culture
Suspension culture eliminates many
disadvantages ascribed to callus culture.
Shaking of cells assures homogenous gaseous
exchange, equal nutrient distribution
(eliminates nutrient gradient within the
medium and within the medium), removes
polarity due to gravity
Cell suspension
culture
 Can be initiated from pre-established callus (large
amounts required 2-3g for 100 cm3) culture or the
explant (takes longer time to establish) on a rotary
shaker. This provides aeration and nutrients and
encourages the callus tissue to break up. Friable
callus preferred but non friable callus can also be
used by changing the media conditions
 Auxins and small amounts of cellulose and pectinase
may be helpful
Cell suspension
culture
Initiation phase: development
of suspension culture from the
callus culture in shaking
conical falsks
Then passed through a nylon
mesh to remove bigger
aggregates
Cell suspension
culture
Filtrate with small cell aggregates
and single cells transferred to fresh
liquid medium for PASSAGE-1
SUSPENSION
By pipetting aliquots into new
media--- usually same conditions
required (lacking Agar) for a specific
species as there growing callus
culture but the conc of auxins and
cytokinins more critical
Cell suspension
culture
Cells have different microenvironment than
free floating cells
1. Have different shapes oval round, elongates,
coiled etc
2. Usually entirely thin walled with few
exceptions that arise as aggregates
3. Suspension cultures may form the whole
plant or embryoids depending upon the
species
Cell suspension
culture
(passage time can be learned
through experience usually
cells 18-25 days for full
density, very active cultures
may take 6-9 days)
Cell suspension
culture
Two types of suspension
culture
1. BATCH CULTURE: Cells grow
in finite volume of agitated
liquid eg. 20-60 ml in conical
flasks incubated on orbital
platform shakers at speed of
0-120 rpm. Same repeated
for subsequent transfers
Cell suspension
culture
 Slow rotating cultures: grown in nipple flasks. 8
nipples– can rotate– each sample bathes and then is
exposed to air at, rotates at 1-2 rpm
 Shake culture: 60-180 rpm– conical flasks circular
motion
 Spinning cultures: 10l bottles, rpm 120 at an angle
of 450 in a culture spinner
 Stirred culture: sample inside a culture medium
displaced by bubbling air. Magnatic stirrer (20600rpm) can be used to agitate culture (1.5-10
liters).
Cell suspension
culture
 CONTINUOUS CULTURE SYSTEM: medium continuously
replaced; replaced when certain nutrient has been
depleted; cell always remain in steady state of active
growth phase
 Chemostats: circular or cylindrical; have inlets and
outlets pores for aeration and introduction and removal
of cells and medium; media stirred by a magnetic
stirrer. Media introduced –equivalent to media dispensed
to compensate for the cells lost during media removal by
new cell division. Can be always maintained at a steady
state– thus density, growth rate, chemical composition
and metabolic activity of cells remain same. Ideal for
studying growth kinetics
Cell suspension
culture
 Turbidostats: can maintain
turbidity and pH of the medium
due to a monitor system that
detects these levels and adds new
media and/or takes out old media.
Introduction into suspension
Sieve out lumps
1
2
Initial high
density
+
Subculture
and sieving
Plate out
Growth kinetics
1. Initial lag dependent on
dilution
2. Exponential phase (dt 1-30
d)
3. Linear/deceleration phase
(declining nutrients)
4. Stationary (nutrients
exhausted)
Large amount of callus is
required.
2-3 gm for 100 cm3
The cells should be
subcultured early during
the stationary phase.
3
2
1
4
Growth kinetics
 Lag phase cells adjust and get ready for cell division
 Log phase cells divide
 Linear phase: Linear increase in number
 Stationary phase: some cells start to die a plateau
 Low or high initial density will ot allow the cells to
grow; Critical initial density CID: cell density
required for cell division (usually 9-15 X 103)
 Growth can be monitored by looking at the cells
under a microscope or in haemocytometer
Test for viability
 Flourescien diacetate stain– dead
cells would fluoresces red
 Evans blue: in viable blue viable
unstained
Reactors for plant
suspension cultures
•
•
•
•
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Modified stirred tank
Air-lift
Air loop
Bubble column
Rotating drum reactor
Synchronization
• Cold treatment: 4oC
• Starvation: deprivation of an
essential growth compound, e.g. N
→accumulation in G1
• Use of DNA synthesis inhibitors:
thymidine, 5-fluorodeoxyuridine,
hydroxyurea
• Colchicine method: arresting the
cells in metaphase stage,
measured in terms of mitotic index
(% cells in the mitotic phase)
Ways to increase product formation
• Select
• Start off with a
producing part
• Modify media for
growth and product
formation.
• Feed precursors or
feed intermediates
(bioconversion)
• Produce ‘plant-like’
conditions
(immobilisation)
Selection Strategies
•
•
•
•
Positive
Negative
Visual
Analytical Screening
Positive selection
• Add into medium a toxic compound e.g.
hydroxy proline, kanamycin
• Only those cells able to grow in the
presence of the selective agent give
colonies
• Plate out and pick off growing colonies.
• Possible to select one colony from millions
of plated cells in a days work.
• Need a strong selection pressure - get
escapes
Negative selection
• Add in an agent that kills dividing cells e.g.
chlorate / BUdR.
• Plate out leave for a suitable time, wash
out agent then put on growth medium.
• All cells growing on selective agent will die
leaving only non-growing cells to now
grow.
• Useful for selecting auxotrophs.
Visual selection
• Only useful for colored or fluorescent
compounds e.g. shikonin, berberine, some
alkaloids
• Plate out at about 50,000 cells per plate
• Pick off colored / fluorescent-expressing
compounds (cell compounds?)
• Possible to screen about 1,000,000 cells
in a days work.
Analytical Screening
• Cut each piece of callus in half
• One half subcultured
• Other half extracted and amount of
compound determined analytically (HPLC/
GCMS/ ELISA)
Metabolites
• Primary metabolites: Molecules that
are essential for growth and
development of an organism.
• Secondary metabolites: molecules
that are not essential for growth and
development of an organism.
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Secondary metabolites are
derived from primary
metabolites
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Why secondary
metabolites?
• Chemical warfare to protect plants
from the attacks by predators,
pathogens, or competitors
• Attract pollinators or seed
dispersal agents
• Important for abiotic stresses
• Medicine
• Industrial additives
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Secondary metabolites
• Possibly over 250,000 secondary
metabolites in plants
• Classified based on common
biosynthetic pathways where a
chemical is derived.
• Four major classes:
Alkaloids, glycosides,
phenolics, terpenoids
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Alkaloids
•
Most are derived from a few
common amino acids (i.e.,
tyrosine, tryptophan, ornithine or
argenine, and lysine)
•
Compounds have a ring structure
and a nitrogen residue.
•
Indole alkaloids is the largest
group in this family, derived from
tryptophan
•
Widely used as medicine
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Phenolics
•
Derived from aromatic amino acids, such as
phenylalanine, tyrosin, and trytophan.
•
All contain structures derived from phenol
•
Some examples:
Coumarins: antimicrobial agents, feeding
deterrents, and germination inhibitors.
Lignin: abundant in secondary cell wall, rigid
and resistant to extraction or many
degradation reagents.
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Terpenoids
•
Terpenes are generally polymers of 5-carbon unit
called isoprene
•
Give scent, flavors, colors, medicine...
•
Three plant hormones are derived from the
terpenoid pathway.
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Glycosides
•
Compounds that contain a carbonhydrate and a
noncarbohydrate
•
Glucosinolates: found primarily in the mustard family
to give the pungent taste.
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Taxol
Taxus brevifolia Nutt.
• Taxol is a terpenoid
• "the best anti-cancer agent” by
National Cancer Institute
• Has remarkable activity against
advanced ovarian and breast
cancer, and has been approved for
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clinical use.
• Camptothecin is an indole alkaloid,
derived from tryptophan.
• Has anticancer and antiviral activity
• Two CPT analogues have been used
in cancer chemotherapy, topotecan
and irinotecan.
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class of alkaloids, the vinca alkaloids from Vinca rosea, the
Madagascar periwinkle, can also bind to tubulin and inhibit
microtubule polymerization. Vinblastine and vincristine are used as
potent agents for cancer chemotherapy,
The alkaloid colchicine, a constituent of the swollen, underground
stems of the autumn crocus (Colchicum autumnale) and meadow
Importance
 Can help study: cell physiology, biochemistry, metabolic
events at the level of single cells or aggregates.
 Help understand the process of organ and embryoid formation
 Single cell clones
 Secondary metabolites
 Mutagenesis to form mutant cell clones. Mutagens such EMS
can be directly added to the medium
 Cell suspension culture techniques are very important for plant
biotransformation and plant genetic engineering.
 Single cells can be placed onto agar medium for formation of
callus and thus forming a whole plant from a single cell
PATHOGEN ELIMINATION
Most plants --- systemic diseases caused by
fungi, viruses bacteria nematodes and
mycoplasmas
• Can lead to death
• Also reduction in yield and quality of crops
• Pathogens nearly always transferred by
vegetative propagation
• Viruses not only in vegetative propagated
organs but also all seeds
PATHOGEN ELIMINATION
 Pathogen elimination desirable:
• Better yield
• Trade across international boundaries
 Can treat fungi with fungicides and
bacteria with bactericides but no
treatment for viruses among plants
 Antimetabolites have helped eradicate
viruses from culture media for lily and
apple
PATHOGEN ELIMINATION
 To eradicate viruses one can select for
heathy plants and propagate them
 For entire population infected with viruses
tissue culture is required
PATHOGEN ELIMINATION
 Apical meristem has no or the least amount of viruses; as
tissues grows older and moves away from meristem the viral
titre also increases.
This may be due to
•
Viruses move through vascular system it is absent in the
meristem
•
High metabolic activity in meristem reduces viral replication
•
High endogenous auxin level may reduce viral multiplication
•
Due to this gradient of viral titer scientists grew meristem and
obtained from it disease free plants
•
Meristem culture also frees from other pathogens
 Plants are free of viruses whose test have been developed
 Well only be virus free if it is free from all known viruses
METHODS FOR VIRAL
ELIMINATION
 HEAT TREATMENT
• Before meristem culture viral eradication
was by heat treatment (thermotherapy)
• Through hot water (dormant buds) or air
(growing shoots) can inactivate viruses
without any harm to the host
• Better host survival in hot air treatment
HOT AIR TREATMENT
•
IN A ATHERMOTHERAPY CHAMBER AT 35-40C from
a few hours to months
•
Requires adequate humidity and light
•
Adequate carbohydrates which can be achieved by
pruning
•
Temperature should be raised gradually over the course
of a few days
•
Small cuttings taken and grafted onto healthy rootstocks
•
Drawbacks: not for all viruses; can be counter
productive by increasing the sensitivity of cured plants
to pathogens over prolonged periods of treatment
•
If some viruses escape this treatment they can be
eradicated by meristem tip culture
MERISTEM TIP-CULTURE
 Size of explant important; if it is too big then it can have
viruses’
 Also in some cases host virus combination also
important
 Same aseptic techniques employed as other cultures
 Meristem tips can be isolated from apices of stems,
tuber sprout, leaf axils, buds of cuttings or germinated
seeds
 My not need surface sterilization as they are protected
very well by leaf primordia; but as a precautionary
measure should be dipped in 75% alcohol
 Surface sterilization required for underground plant
organs
MERISTEM TIP-CULTURE
 As explant is very small a stereoscopic
microscope should be used and care
should be taken to not desiccate the
meristem
 Can perform this excision over a petridish lined with sterile filter paper
 Instruments need to be sterilized
MERISTEM TIP-CULTURE
Method
1. Hold bud under microscope with a forcep
2. Leaves and leaf primordia are removed
by needle to expose apical meristem (A
shiny dome)
3. Excise meristem by a clean cut with a
sharp blade
4. Same instruments can be used to transfer
the tip to culture media
MERISTEM TIP-CULTURE
Physiological condition of the explant:
•
Should take actively growing bud; so position from
where taken important. In some cases terminal buds
better than auxiliary buds in other cases no difference.
•
Season or time of year also important; should be taken
when active not dormant or after dormancy has been
broken with suitable treatment
•
More axillary buds can be used to get better escape
from viral titer
•
Should also be able to root and shoot so need to keep
those requirements in mind
MERISTEM TIP-CULTURE
AND THERMOTHERAPY
• Some viruses present in the meristem
such as TMV; both treatments effective in
getting rid of viruses under such
circumstances
• Heat treatment can be given to mother
plant as well as the meristem culture
• Can alternate heat and normal
temperatures through out the day to avoid
damage to the explant or deterioration of
meristem tip culture
Culture media
• Small amounts of auxins and cytokinins
have been useful in the MS media
• GA3 in some cases can also be added
• Both agar and liquid media have been
used but agar mostly preferred
• In case of liquid media the explant can be
placed on a filter paper dipped at both
sides into the media and the explant
resting above the media
STORAGE CONDITIONS
• Need adequate light except those cultures
that need to be grown in the dark to
minimize production of polyphenols
• Light can be increased as the explant
grows after a suitable period of time
CHEMICAL TREATMENT
• Not very useful and shows conflicting
results
• Antimetabolites had little effect (malachite
green)
• Nucleoside analogue ribavirin removed
PXC virus in potato, CMV and alfalfa
mosaic virus in other cultures
• Vidarbine, cycloheximide and actinomycin
D have aslo been useful
OTHER METHODS (in vivo)
•
In callus cultures distribution of viruses is uneven; some
cells don’s have them
•
This is because:
1. Virus replication cannot keep pace with the dividing
cells
2. Cells acquire resistance to viruses through
mutagenesis
3. Some drawbacks include: 1: Genetic instability and 2:
low potential of plant regeneration
4. Other methods include somatic cell hybridization, gene
transformation and somaclonal variations
VIRUS INDEXING
• Used to determine if viruses really have
been eradicated; need to do several times
during the plant growth period as some
viruses may sprout later. A consistent
negative result will only show that viruses
are not present:
VIRUS INDEXING: METHODS
USED
•
Sap transmission test:
Most sensitive of all three methods
Leaves taken and ground in a pestle and mortar with equal
volume of buffer. Then sprayed on an indicator plant (Plant
susceptible to the virus), if plant develops symptoms then
virus present
•
Serology: a drop of sap from the test plant can be
added an antiserum solution obtained from rabbits with
the specific antibodies; precipitation indicated a reaction
between the viral proteins and the antibodies (ELISA)
the enzyme linked immunosorbant assay can be used
for this purpose
•
EM STUDIES:
MICROGRAFTING
• Initiating meristem cultures from woody
plants is very difficult
• So can do micro grafting
• Can take meristem and graft it onto a
virus free rootstock (seedling) maintained
and propagated in vitro.
• Specially useful in eliminating viruses
from fruit crops
Eradicating other pathogens
• Fungi, bacteria and mycoplasmas can
also be removed by using meristem-tip
culture and callus culture
PROTOPLAST
CULTURE
1. Introduction
The term protoplast was introduced by Hanstein in 1880.
It refers to the cellular content excluding cell wall or
can also be called as naked plant cell. It is described
as living matter enclosed by a plant cell membrane.
Protoplast isolation for the first time was carried out by
Klercker in 1892 using mechanical method on the
plasmolysed cells.
The application of protoplast technology for the
improvement of plants offers fascinating option to
complement conventional breeding programs.
The ability of isolated protoplasts to undergo fusion and
take up macromolecules and cell organelles offers
many possibilities in genetic engineering and crop
improvement.
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The experiments involving protoplasts consist
of three stages –
i. protoplast isolation
ii. protoplast fusion (leading to gene uptake)
iii. development of regenerated fertile plants from
the fusion product (Hybrid).
Depending upon the species and culture
conditions, the protoplasts may have the
potential to:
i. regenerate a cell wall
ii. dedifferentiate to form callus
iii. divide mitotically and proliferate clonally
iv redifferentiate into shoots, roots or embryos 70
and produce a complete plantlet.
However, to fully explore the potentials for
protoplast-technology,
efficient
and
reproducible
methods
for
protoplast
isolation and purification must first be
established.
Since leaf tissue is a readily accessible source
of genetically uniform cells, it is often
desirable to use mesophyll protoplasts in
somatic hybridization studies, but, leaf
tissues, in general, do not yield large number
of protoplasts owing to the difficulty in
removing the lower epidermis.
An alternative, therefore, is the cultured cell
material where protoplasts can show greater
potential to divide.
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2. Protoplast isolation
Protoplast isolation may be carried out by
1. Mechanical disruption method or
2. Enzymatic method.
* NOTE:
Out of these two methods, enzymatic
method is preferred as it provides better
protoplast yield with low tissue damage
while
mechanical
method
causes
maximum tissue chopping with lower
protoplast yields.
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i. Mechanical method
Klercker in 1892 pioneered the isolation of protoplasts by
mechanical methods.
In this method, the cells were kept in suitable plasmolyticum, for
example CPW containing 13% w/v mannitol.
Once the plasmolysis is complete, while remaining in the
osmoticum, the leaf lamina would be cut with a sharp-edged
knife. In this process some of the plasmolyzed cells were cut
only through the cell wall, releasing intact protoplasts while
some of the protoplasts may be damaged inside many cells.
Protoplasts that were trapped in a cell and only the corner had
been cut off could be encouraged to come out by reducing the
osmolarity slightly to force the protoplasts swell to force their
way out of the cut surface.
The released protoplasts then have to be separated from
damaged ones and cell debris.
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Disadvantages
•
•
•
•
Lower protoplast yield.
Labour intensive method.
Protoplast obtained has low viability.
Method is applicable only to vacuolated cells.
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ii. Enzymatic method
In
1960, E.C.Cocking demonstrated the
possibility of enzymatic isolation of a large
number of protoplasts from cells of higher
plants.
This method involves leaf sterilization followed
by peeling of the lower epidermis to release
cells which are plasmolyzed and added to
enzyme mixture followed by harvest of
protoplast as shown in (Figure).
Either of the procedures for enzymatic isolation
can be used: sequential enzymatic hydrolysis
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or mixed enzymatic hydrolysis.
A. SEQUENTIAL ISOLATION
In the sequential isolation,
1. Firstly, cells are separated by the use of a
maceration enzyme – a pectin hydrolyzing
enzyme such as, macerozyme or Pectolyase.
2. Once the cells are separated, they are
washed in CPW solution free of enzymes but
containing
plasmolyticum
by
gentle
centrifugation (100g).
3. The pellet is retained and resuspended in the
second
enzyme
like,
cellulases
and
hemicellulases, used to hydrolyse the
remaining cell was component.
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4. Once the protoplasts are released they are
washed with CPW to remove the debris.
B. MIXED ENZYMATIC APPROACH
In the mixed enzymatic approach, Plant
tissues are plasmolyzed in the presence
of a mixture of pectinases and cellulases,
thus, inducing simultaneous separation of
cells and degradation of their walls to
release the protoplasts directly in a single
step.
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Figure : Steps involved in protoplast isolation, fusion and regeneration
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CONDITIONS REQUIRED FOR ENZYME
ACTIVITY
Enzymes are pH and temperature dependent, thus,
for enzymatic release of protoplast an enzyme
showing activity at pH range 4.7-6.0 and
temperature range of 25-30°C is used.
• Duration of enzyme pretreatment and condition of
light presence required for incubation may also be
determined.
• Enzyme mixture used should essentially consist of
cellulase, hemicellulase and pectinase which
facilitate
the
degradation
of
cellulose,
hemicelluloses and pectin, respectively.
• The concentration of sugar alcohols used 79as
osmoticum (mannitol) must be empirically defined.
Factors affecting yield and
viability of protoplasts
1. Source of material
2. Pre-enzyme treatments
3. Enzyme treatment
80
1. SOURCE OF MATERIAL
Leaves were the most convenient source of the plant protoplasts
because it allows
1. The isolation of a large number of relatively uniform cells
without killing the plants.
2. Moreover the mesophyll cells are loosely arranged, the
enzymes have an easy access to the cell wall.
3. The parent plant age and the conditions in which it is growing
have profound effect on the yield of protoplast.
4. Due to the difficulty in isolating culturable protoplast from leaf
cells of cereals and some other species their cultured cells
can be used as a source material.
5. The yield of protoplasts depends upon the growth rate and
growth phase of the cells.
6. Generally embryogenic suspension cultures are used to obtain
totipotent protoplasts .
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2. PRE-ENZYME TREATMENTS
To facilitate the penetration of enzyme solution into the
intercellular spaces of leaf, which is essential for effective
digestion, various methods are followed.
1. The most commonly used method is to peel the lower
epidermis and float the stripped pieces of leaf on the enzyme
solution in a manner that the peeled surface is in a contact
with the solution.
2. Most of the time it is not convenient to peel the epidermis, in
such cases cutting the leaf or tissue into small strips (1- 2 mm
wide) has been found useful. When combined with vacuum
infiltration the latter approach has proved very effective.
4. Brushing of leaves with a soft brush or with the cutting edge
of a scalpel may also improve the enzymatic action.
5. Large calli are chopped into pieces and can be transferred to
enzyme mixture.
6. Agitation of incubation mixture during enzyme treatment
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improves protoplast yield from cultured cells.
3. ENZYME TREATMENT
 The release of protoplast is very much dependent
on the nature and concentration of the enzymes
used.
 The two major enzymes required for the isolation
of protoplast are cellulase and pectinase.
 The cellulase is required to digest the cellulosic
cell walls and the pectinase mainly degrades the
middle lamella.
 Some of the tissues may require other enzymes
like, hemicellulase, driselase, macerozyme and
pectolyase.
 The activity of enzyme is pH dependent. The pH of
the enzyme solution is adjusted somewhere
83
between 4.7 to 6.0.
CHOICE OF PLASMOLYTICUM
The two most commonly used compounds are the
sugar alcohols - mannitol and sorbitol.
Of these, mannitol is the most preferred since it is
not metabolized by the plant cells.
Once the protoplast divides and regenerates the cell
wall, no more osmoticum is required. It is,
therefore, should be removed gradually from the
medium otherwise cell division stops.
To slowly remove the osmoticum from the medium,
the protoplast can be isolated in a high osmoticum
mixture consisting of both mannitol and sucrose,
the sucrose will be metabolized by the dividing
protoplasts and thus, will reduce the osmolarity of
the medium. Normally, mannitol is used84 at
concentration range of 11-13%.
Cocking, Peberdy and White –
CPW
A solution into which the osmoticum is often, but
not always, added is called CPW salts mix or CPW
for short. This has been observed much more
beneficial than using distilled water as a solvent
in obtaining high yields of viable protoplasts.
Although CPW is most widely used solution into
which osmoticum or enzymes are added, some
times culture medium used to grow cells or plants
can also be utilized for protoplast isolation at one
tenth concentration.
Low concentration of culture medium is much more
advantageous when compared with CPW.
85
Table: Salt mix of protoplast washing media solution
(Cocking, Peberdy and White – CPW)
86
PROTOPLAST PURIFICATION
Enzyme treatment results in suspension of protoplast,
undigested tissues and cellular debris.
This suspension is passed through a metal sieve or a
nylon mesh (50-100 µm) in order to remove undigested
cellular clumps.
The filtered protoplast-enzyme solution is mixed with a
suitable volume of osmoticum, solution is centrifuged to
pellet the protoplasts, pellet of protoplast is resuspended
in osmoticum of similar concentration as used in enzyme
mixture.
The protoplast band is sucked in Pasteur pipette and is put
into other centrifuge and finally suspended in culture
medium at particular density; this is explained by 87
the
Figure.
88
PROTOPLAST VIABILITY
The isolated protoplast must have a spherical shape when observed by
a light microscope, protoplast can be stained using following stain:
1. Fluorescein diacetate staining method: FDA accumulates inside the
plasmalemma of viable protoplasts. Live protoplasts contain
esterases which cleave FDA to release fluorescein which fluoresces
yellowish-green using fluorescence microscopy within 5 min. FDA
dissociates from membrane after about 15 min. It is used at a
concentration of 0.01% dissolved in acetone.
2. Calcofluor White staining: This staining method assures protoplast
viability by detecting onset of cell wall formation. Calcofluor binds to
beta linked glucosides in newly synthesized cell wall which can be
observed as a fluorescent ring around the membrane. Optimum
staining is achieved when 0.1 ml of protoplast is mixed with 5.0 μl of
0.1% w/v solution of CFW.
3. Protoplast viability can also be detected by monitoring oxygen
uptake of cells by oxygen electrode, which shows respiration.
4. Variation of protoplast size with changing osmotic concentration
also enables viability of protoplast.
89
3. PROTOPLAST CULTURE
Protoplasts culture techniques
The culture requirements and the culture
methods are same for both protoplasts and
single cells.
The main difference is the requirement of
suitable osmoticum for protoplasts until they
regenerate a strong wall.
Isolated protoplasts are either cultured in liquid
or semisolid agar or agarose media plates,
sometimes the protoplast is first grown in
liquid media and then transferred into the
agar media plates.
90
The following techniques have been adopted in
order to maintain number of protoplast
population between minimum and maximum
effective densities after plating up:
i. Liquid method
ii. Embedded in Agar/ Agarose
iii. Feeder layer
iv. Co-culturing
91
i. Liquid method
This method is preferred in earlier stages of
culture as it provides
(a) easier dilution and transfer,
(b) the osmotic pressure of liquid media can be
effectively reduced after a few days of culture
(c) the cell density can be reduced or cells of special
interest can be isolated easily.
In Liquid medium, the protoplast suspension is
plated as a thin layer in petriplates, incubated
as static culture in flasks or distributed in 50100 μl drops in petriplates and stored in a
92
humidifier chamber.
ii. Embedded in Agar/ Agarose
Agarose is a preferred choice in place of agar and this has
improved the culture response.
This method of agar culture keeps protoplast in fixed position,
thus, prevents it from forming clumps.
Immobilized protoplasts give rise to clones which can then be
transferred to other media.
In practice, the protoplasts suspended in molten (40°C) agarose
medium (1.2% w/v agarose) are dispensed (4ml) into small
(3.5-5cm diameter) plates and allowed to solidify.
The agarose layer is then cut into 4 equal sized blocks and
transferred to larger dishes (9 cm diameter) containing liquid
medium of otherwise the same composition.
Alternatively, protoplasts in molten agarose medium are
dispensed as droplets (50-100 μl) on the bottom of petri plates
and after solidification the droplets are submerged in the same
liquid medium.
93
iii. Feeder layer
In order to culture protoplast at low density,
a feeder layer technique is adopted.
A feeder layer of X-ray irradiated nondividing
but
metabolically
active
protoplasts after washing are plated in soft
agar medium.
Non-irradiated protoplasts of low density are
plated over this feeder layer.
The protoplasts of the same species or
different species can be used as a feeder
94
layer.
iv. Co-culturing
This method involves co-culture of protoplasts from
two different species to promote their growth or
that of the hybrid cells.
Metabolically active and dividing protoplasts of two
types - slow and fast growing are cultured together,
the fast growing protoplast provide other species
with diffusible chemicals and growth factors which
helps in cell wall formation and cell division.
The co-culture methods is generally used where calli
arising from two types of protoplasts can be
morphologically distinguished.
For example, protoplasts isolated from albino plants
and green plants are easily distinguishable based
on color where albino protoplast will develop 95
non
green colonies.
96
Figure: Protoplast culture techniques
CULTURE MEDIUM
The nutritional requirement of protoplast is almost
similar to that of the cultured plant cells.
Mostly the salts of MS (Murashige and Skoog, 1962) and
B5 (Gamborg et al. 1968) media and their modifications
have been used.
Ammonium salts have been found detrimental to
protoplasts survival of many species, and media have
been devised that either have a reduced concentration
of ammonium or lack it.
Concentration of zinc is reduced while the concentration
of calcium is increased as it enhances the membrane
stability.
Osmolarity is maintained by addition of sorbitol,
mannitol, glucose or sucrose and mannitol being
widely used osmoticum as it is not used by 97the
dividing cells, thus, maintains the osmolarity of the
medium.
Glucose is preferred carbon source as sucrose
do not satisfy protoplast culture. One or two
amino acids are added at low concentration.
Growth regulators are required essentially in
protoplast
culture
generally
high
concentration of auxins (NAA, 2,4-D) along
with lower concentration of cytokinins (BAP,
Zeatin ) is used.
Environmental conditions: High light intensity
inhibits growth of protoplast hence initially
protoplast is grown in dim light for few days
and then transferred to light of about 20005000 lux. However, better results are obtained
when cultured in darkness.
98
Plating density
Like cell cultures, the initial plating density of
protoplasts has profound effect on plating
efficiency.
Protoplasts are cultured at a density of 1 x 104 to 1 x
105 protoplasts ml-1 of the medium.
At high density the cell colonies arising from
individual protoplasts tend to grow into each other
resulting into chimera tissue if the the protoplast
population is genetically heterogeneous.
Cloning of individuals cells, which is desirable in
somatic hybridization and mutagenic studies, can
be achieved if protoplasts or cells derived from
99
them can be cultured at a low density.
4. Protoplast development and regeneration
Protoplast starts to regenerate a cell wall within few days
(2-4 days) of culture and during this process,
protoplasts lose their characteristic spherical shape
which has been taken as an indication of new wall
regeneration.
Cell wall regeneration can be confirmed by Calcofluor
White staining method. There is direct relationship
between wall formation and cell division. Protoplasts
which are not able to regenerate a proper wall fail to
undergo normal mitosis.
Protoplasts with a poorly developed wall often show
budding and may enlarge several times their original
volume. They may become multinucleate because
karyokinesis is not accompanied by cytokinesis.
Among other reasons, inadequate washing of the
protoplasts prior to culture leads to these
100
abnormalities.
Figure : Protoplast isolation and cell wall regeneration. A. Isolated
protoplast showing spherical structure; B. Wall is regenerated around the
protoplast and one of the protoplasts showing cell division (arrow marked)
101
A healthy 4-week-old Arabidopsis plant suitable for protoplast isolation.
102
103
Using Tape-Arabidopsis Sandwich for Arabidopsis protoplast
isolation
104
105
106
Figure 1. Isolation of protoplasts from Arabidopsis seedlings. A, Tissues from 14d-old seedlings of Arabidopsis were collected and converted to protoplasts by a
modified procedure of Chen and Halkier (2000). B, Protoplasts were purified by
Suc density gradient centrifugation and collected at the interface of enzyme
solution and W5 buffer (white arrows). C, Bright-field microscopy of protoplasts.
107the
Microphotographs were collected using a cooled CCD camera interfaced with
Zeiss Axioscope 2 plus microscope.
And completes process when provided with
suitable condition of light, pH and temperature
newly synthesized protoplast can be
visualized by staining.
Once the cell wall formation is completed, cells
undergo division resulting in increase size of
cells.
After an interval of 3 weeks, small cell colonies
appear, these colonies are transferred to an
osmotic-free callus induction medium.
This is followed by introduction into
organogenic or embryogenic medium leading
108
to plantlet development.
SOMATIC HYBRIDIZATION
Development of hybrid plants through
the fusion of somatic protoplasts of two
different plant species/varieties is called
somatic hybridization
HISTORY
The fusion of plant protoplasts is not
a particularly new phenomenon.
Kuster in 1909 described the process
of random fusion in mechanically
isolated protoplasts.
FUSION PRODUCTS
Cells containing nonidentical nuclei are
refered to as “heterokaryons” or
“heterokaryocytes”.
The fusion of nuclei in a binucleate
heterokaryon results in the formation of
a true hybrid protoplast or
“synkaryocyte”.
The fusion of two protoplasts from the
same culture results in a
“homokaryon”.
Frequently genetic information is lost
from one of the nuclei. If one nucleus
completely disappears, the cytoplasm
of the two parental protoplasts are still
hybridized, and the fusion product is
known as a “cybrid” (cytoplasmic
hybrid) or “ heteroplast”.
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
Isolation of Protoplast
(Separartion of protoplasts from plant tissue)
1. Mechanical
Method
2. Enzymatic
Method
1. Mechanical Method
Cells Plasmolysis
Plant Tissue
Microscope Observation of cells
Release of protoplasm
Cutting cell wall with knife
Collection of protoplasm
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
Enzymatic Method
Leaf sterlization, removal of
epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase
Pectinase +cellulase
Protoplasm released
Isolated
Protoplasm
Release of
isolated cells
cellulase
Protoplasm
released
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
Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous
Fusion
Intraspecific
Intergeneric
2. Induced
Fusion
Chemofusion
Mechanical
Fusion
Electrofusion
Spontaneous Fusion
Protoplast fuse spontaneously during
isolation process mainly due to physical
contact
• Intraspecific produce homokaryones
• Intergeneric have no importance
Induced Fusion
Chemofusion- fusion induced by chemicals
• Types of fusogens
• PEG
• NaNo3
• Ca 2+ ions
• Polyvinyl alcohol
Induced Fusion
Mechanical Fusion- Physical fusion of
protoplasts under microscope by using
micromanipulator
and
perfusion
micropipette.
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
microseconds.
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
• Presence of chloroplast
• Nuclear staining
• Heterokaryon is stained by carbol-fuschin,
aceto-carmine or aceto-orcein stain
Hybrid Selection
(Several markers are used )
• Genetic complementation
• Phytotoxins
• Specific amino acid
• Auxin autotrophy
• Antibiotics
• Auxotrophic and metabolic mutants
• Chromosomal analysis
• Herbicides
Culture of the hybrid cells
Hybrid cells are cultured on
suitable medium provided with
the appropriate culture
conditions.
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.
Advantages of somatic
hybridization
Production
of
novel
intergenic hybrid
interspecific
and
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
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
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