Tissue Culture Techniques with the Modern Crop Improvement Trends

1. Use of Tissue Culture for crop
improvement and protection
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2. SMALL PIECES OF LIVING TISSUE / PLANT
PARTS- Explants
GROWN UNDER STERILE IN VITRO
CONDITIONS
ON ARTIFICIAL NUTRIENT MEDIA (defined or
semi defined)
FOR INDEFINITE PERIODS OF TIME
TISSUE CULTURE
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Principles Involved
•VERY SIMPLE!!!
•Isolate a plant part
•Provide appropriate conditions

4. TOTIPOTENCY OF PLANT CELLS
• Totipotency is the ability of a single cell to divide
and produce all the differentiated cells in an
organism.
•Every cell of a plant has the potential to grow into a
complete plant
•Tissue culture techniques are based on this
potential/ totipotency of cells
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6. SELECT EXPLANT
This can be any part of the plant or any appropriate tissue
TRIMMING
Trim to a suitable size
SURFACE STERILISATION
For the removal of surface contaminants
GENERALLISED PROCEDURE
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7. SERIAL WASHING
For the removal of surface steriliant
FINAL TRIMMING
To remove tissue penetrated by the surface sterilising agent
CULTURE ESTABLISHMENT
Introduction into the suitable medium
INCUBATION
Suitable conditions- TEMPERATURE, LIGHT INTENSITY AND DURATION
SUB CULTURE
Due to limitation in nutrients, space etc
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8. • TO REMOVE SURFACE CONTAMINANTS
• WHY?
• CHEMICALS USED:
Tap water
Ethyl alcohol
Sodium hypochlorite
Calcium hypochlorite
Bromine water
Mercuric chloride
SURFACE STERILISATION
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9. • MULTI -FACTORIAL EXPERIMENT
• RANGE OF CONCENTRATIONS
• RANGE OF TIME INTERVALS
• TEST FOR VIABILITY
Using a vital stain
• TEST FOR STERILITY
Plating in culture media for microorganisms
LOWEST CONCENTRATION AND TIME DURATION SELECTED AS SUITABLE
THE CONCENTRATION AND TIME DURATION- How
do we know?
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10. • MULTI -FACTORIAL EXPERIMENT
• RANGE OF CONCENTRATIONS
• RANGE OF TIME INTERVALS
• TEST FOR VIABILITY
Using a vital stain
• TEST FOR STERILITY
Plating in culture media for microorganisms
LOWEST CONCENTRATION AND TIME DURATION SELECTED AS SUITABLE
THE CONCENTRATION AND TIME DURATION- How
do we know?
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11. Wash with running tap water
Wash with 70% ethanol
Immerse in appropriate chemical
▪ Appropriate concentration for
▪ Appropriate time duration
Surface Sterilisation- PROCEDURE
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12. Murashige and Skoog medium (MS medium)
Gamborg medium
White’s medium
CULTURE MEDIA
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COMPONENTS OF CULTURE MEDIA
ORGANIC AND INORGANIC COMPONENTS NEEDED FOR PLANT GROWTH
• MACRO AND MICRO ELEMENTS
MACRO- C H O N P K Ca Mg S
MICRO- Fe Mn Zn B Al Cl Na
• CARBON SOURCE- SUCROSE, GLUCOSE etc
• VITAMINS AND OTHER GROWTH FACTORS THIAMINE, NICOTINIC ACID, ASCORBIC ACID, INOSITOL, AMINO
ACIDS
• GROWTH REGULATORS- AUXINS, CYTOKININS, GIBBERELLINS
• DISTILLED/DEIONISED WATER
pH- 5.7-5.8

14. MS Medium reciepe
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15. STERILISATION METHODS
Dry heat- glassware, metal instruments
Oven at 1600 c for 4 hrs
Wet heat- media, distilled water
Autoclave/ pressure cooker (15 lb/in2 pressure, 15 minutes)
Ultra -filtration- for media components that get destroyed by
heat
Ultra filters (0.22µm)
Chemicals- working surfaces, explants etc
Ethanol (70%, 90%), Sodium/calcium hypochloride,
bromine water etc
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17. Laboratory Design and Development
A standard tissue culture laboratory should provide facilities for:
• washing and storage of glassware, plastic ware
• preparation, sterilization and storage of nutrient media
• aseptic manipulation of plant material
• maintenance of cultures under controlled temperature, light and humidity
• observation of cultures, data collection and photographic facility
• acclimatization of in vitro developed plants.
The overall design must focus on maintaining aseptic conditions.
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18. Ideal tissue culture facility
At least three separate rooms should be available:
• one for washing up, storage and media preparation -
preparation room
• a second room, containing laminar-air-flow or clean air
cabinets for dissection of plant tissues and subculturing –
culture room
• and the third room to incubate cultures- growth room
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• Washing glassware
• Media preparation
• Sterilisation of media, glassware etc
• 6x4.5 m
• Proper ventilation
• Distilled water supply
Preparation room

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• Excision/isolation of explant
• Trimming
• Surface sterilisation
• Inoculation to culture media
• 3x4.5 m
• Filtered air (filter on the air conditioner)
• Smooth walls, roof etc
• Laminar flow cabinet can be used instead
Culture Room

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• Incubation of cultures
• Controlled temperature
• Controlled light
light intensity
duration of day length
• 6x4.5 m
• No ventilation from outside
• Shelves for keeping solid cultures
• Shaker(s) for liquid cultures- Cell suspension, protoplast cultures
Growth Room

23. Growth Room
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24. • Organ culture- roots, shoot tips, nodes, petioles,
pollen and anther, flowers, embryos etc
• Callus culture
• Cell suspension culture
• Protoplast culture
TYPES OF CULTURES
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25. • Organ culture- roots, shoot tips, nodes, petioles,
pollen and anther, flowers, embryos etc
• Callus culture
• Cell suspension culture
• Protoplast culture
TYPES OF CULTURES
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27. Surface sterilise unopened flowers
Excise anthers or pollen
Grow on nutrient media
Anther culture
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28. Shoot tip culture
The apical meristem of a shoot is the
portion lying distal to the youngest
leaf primordium
Measures up to about 100 µm in
diameter and 250 µm in length. The
apical meristem together with one to
three young leaf primordia,
measuring 100-500 µm, constitutes
the shoot-apex (Figure A and B)
Used in virus elimination
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31. Nodal Culture
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32. Genetically identical plants/ less or no
mutations
Clones of the mother plant
Low production rate
Labour/ time consuming
Advantages and disadvantages
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33. What is a callus culture?
Growing and dividing mass of cells.
Callus culture
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34. Select explant
Surface sterilise
Culture in suitable medium
Incubate under appropriate conditions
Transfer the calli into fresh media
Subculture
Initiation and maintenance of callus cultures
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36. Friable- loosely packed unorganised cells
Organogenic- give rise to organs
Embryogenic- give rise to embryos
Both contain organised compact cells
Callus types
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Growth Kinetics

38. Organogenic callus
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39. • naked embryos can be converted to ‘synthetic seeds' or ‘syn
seeds' for large scale clonal propagation at commercial level.
• This can be achieved by encapsulating the viable somatic
embryos in a protective covering.
Production of synthetic seeds or artificial seeds
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40. Somaclonal variation
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Methods of producing Somaclonal variants

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49. Cell suspension cultures- Growth
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• Number of cells:
• Fresh weight of cells
• Dry weight of cells
• Number of viable cells
• Packed Cell Volume (PCV)
Determination of sub culture time of a cell suspension culture

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• Paper raft nurse technique
• Petri dish plating technique
• Micro-chamber technique
Methods of culturing single cells

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• Single cells are isolated from suspension cultures or a friable callus with the help
of a micropipette or micro-spatula.
• Few days before cell isolation, sterile 8 mm x 8 mm squares of filter paper are
placed aseptically on the upper surface of the actively growing callus tissue of
the same or different species.
• The filter paper will be wetted by soaking the water and nutrient from the callus
tissue.
• The isolated single cell is placed aseptically on the wet filter paper raft.
• The whole culture system is incubated under 16 hrs. cool white light (3,000 lux)
or under appropriate light and temperature
• The single cell divides and re-divides and ultimately forms a small cell colony.
• When the cell colony reaches a suitable size, it is transferred to fresh medium
where it gives rise to the single cell clone
PAPER RAFT NURSE TECHNIQUE

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PETRI DISH PLATING TECHNIQUE:

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• A drop of the medium carrying a single cell is isolated from suspension cultures, placed on a
sterile microscope slide and ringed with sterile mineral oil.
• A drop of oil is placed on either side of the culture drop and coverslip raisers placed on each
drop.
• A third coverslip is then placed on the culture drop bridging the two coverslips and forming a
microchamber to enclose the single cell aseptically within the mineral oil.
• The oil prevents water loss from the chamber but permits gaseous exchange.
• The whole microchamber slide is placed in a petri-dish and incubated.
• When the cell colony becomes sufficiently large the coverglass is removed andthe tissue is
transferred to fresh liquid or semi-solid medium.
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MICRO-CHAMBER TECHNIQUE

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MICRO-CHAMBER TECHNIQUE

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Protoplast Fusion

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• Number of cells:
• Fresh weight of cells
• Dry weight of cells
• Number of viable cells
• Packed Cell Volume (PCV)
Determination of sub culture time of a cell suspension culture

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• Paper raft nurse technique
• Petri dish plating technique
• Micro-chamber technique
Methods of culturing single cells

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• Single cells are isolated from suspension cultures or a friable callus with the help
of a micropipette or micro-spatula.
• Few days before cell isolation, sterile 8 mm x 8 mm squares of filter paper are
placed aseptically on the upper surface of the actively growing callus tissue of
the same or different species.
• The filter paper will be wetted by soaking the water and nutrient from the callus
tissue.
• The isolated single cell is placed aseptically on the wet filter paper raft.
• The whole culture system is incubated under 16 hrs. cool white light (3,000 lux)
or under appropriate light and temperature
• The single cell divides and re-divides and ultimately forms a small cell colony.
• When the cell colony reaches a suitable size, it is transferred to fresh medium
where it gives rise to the single cell clone
PAPER RAFT NURSE TECHNIQUE

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PETRI DISH PLATING TECHNIQUE:

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• A drop of the medium carrying a single cell is isolated from suspension cultures, placed on a
sterile microscope slide and ringed with sterile mineral oil.
• A drop of oil is placed on either side of the culture drop and coverslip raisers placed on each
drop.
• A third coverslip is then placed on the culture drop bridging the two coverslips and forming a
microchamber to enclose the single cell aseptically within the mineral oil.
• The oil prevents water loss from the chamber but permits gaseous exchange.
• The whole microchamber slide is placed in a petri-dish and incubated.
• When the cell colony becomes sufficiently large the coverglass is removed andthe tissue is
transferred to fresh liquid or semi-solid medium.
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MICRO-CHAMBER TECHNIQUE

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MICRO-CHAMBER TECHNIQUE

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Protoplast Fusion

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• Spontaneous fusion is of no value as fusion of protoplasts of
different origins is required in somatic hybridization.
• To achieve this, a suitable agent (fusogen) is added to fuse
the plant protoplasts of different origins.
• The different fusogens employed are: NaNO3, artificial sea
water, lysozyme, high pH/Ca++, polyethylene glycol,
antibodies, concavalin A, polyvinyl alcohol, electrofusion
dextran and dextran sulphate, fatty acids and esters.
Induced fusion methods

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• Kao and Michayluk (1974) and Wallin et al. (1974)
• The protoplasts are suspended in a solution containing high
molecular weight PEG, which improves agglutination and fusion of
protoplasts in several species.
• 1 ml of the protoplasts suspended in a culture medium are mixed
with 1 ml of 28–56% PEG (1500 –6000 MW) solution.
• The tube is then shaken for 5 sec and allowed to settle for 10 min.
• To remove PEG, the protoplasts are then washed several times by the
addition of protoplast culture medium.
• The protoplast preparation is again suspended in the culture medium.
Polyethylene glycol (PEG) method

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• The PEG method is popular for protoplast fusion as it yields in reproducible high-
frequency heterokaryon formation, low cytotoxicity to most cell types and the
formation of binucleate heterokaryons.
• PEG-induced fusion is non- specific and is thus applicable for interspecific,
intergeneric or interkingdom fusions.
• Both the molecular weight and the concentration of PEG are critical in inducing
successful fusions.
• PEG less than 100 molecular weight is not able to produce tight adhesions while
that ranging up to 6000 molecular weight can be more effective per mole in
inducing fusions.
• At higher molecular weight PEG produces too viscous a solution which cannot be
handled properly.
• Treatment with PEG in the presence of/or by high pH/Ca++ is reported to be most
effective in enhancing the fusion frequency and survivability of protoplasts.
Advantages

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• Protoplasts are placed in to a small culture cell containing electrodes, and a potential difference is
applied due to which protoplasts line up between the electrodes.
• If now an extremely short wave electric shock is applied, protoplasts can be induced to fuse.
• In this fusion method, two-step procedure is followed beginning with application of an alternating
current (AC) of low intensity to protoplast suspension.
• Dielectrophoretic collectors adjusted to 1.5 V and 1 MHz and an electrical conductivity of the
suspension medium less than 10–5 sec/cm generate an electrophoresis effect that make the cells
attach to each other along the field lines.
• The second step of injection of an electric direct current (DC) field pulse of high intensity (750–
1000 V/cm) for a short duration of 20–50 μsec leads to breakdown of membranes in contact areas
of adjacent cells resulting in fusion and consequent membrane reorganization.
• simple, quick and efficient. Cells after electrofusion do not show cytotoxic response.
• specialized equipment is required.
Electrofusion

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Microinjection

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Gene Gun