Tissue culturing
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Transcript of Tissue culturing
Tissue Culture Technique
What is Tissue Culture Technique?
Tissue culture is a process that involves exposing plant tissue to a specific regimen of
nutrients, hormones, and light under sterile, in vitro conditions to produce many new plants,
each a clone of the original mother plant, over a very short period of time.
HistoryHaberland (1902) attempted to culture
isolated mesophyll cells but not succeeded.Guatheret (1939) cultured callus of carrot.Miller (1957) put forth the Hormone
hypothesisS.G. Guha (1966) cultured pollens to obtain
haploid plant.A.F. Mascarens (1991) induced flowering in
bamboo plant by tissue culture technique
Basic concepts of plant tissue culture(PTC)
Two concepts1) Plasticity.
-ability to initiate cell division from almost any tissue of the plant.
-ability to regenerate lost organs.
2) Totipotency. -each cell has the capacity to regenerate the
entire plant.
Basic concepts of plant tissue culture(PTC)
Cell lines differentiate to form specialized tissues and organs.
Unlike animal cells, living plant cells re-differentiate.
Therefore, tissue can be regenerated from explants such as leaf, ovary, protoplast, petiole, root, anthers, etc.
Factors affecting Tissue Culture
Growth Media– Minerals, Growth factors, Carbon source, Hormones
Environmental Factors– Light, Temperature, Photoperiod.
Explant Source– Usually, the younger, less differentiated plant is better for tissue culture
Genetics– Different species show different ability in tissue culture. In many cases, different genotypes within a species will have variable responses to tissue culture.
three main steps to the tissue culture
process: STAGE I is the initiation phase. It concerns the establishment of plant tissue in vitro by sterilizing the
material and initiating it into culture. STAGE II is the multiplication phase. At this stage, the in
vitro plant material is re-divided and placed in a medium with plant growth regulators that induce the proliferation of multiple shoots. This process is repeated many times
until the number of plants desired is reached. STAGE III is the root formation phase. It involves the
introduction of hormones to induce rooting and the formation of complete plantlets.
Types of tissue culturePlant tissue culture(Micropropagation). Animal tissue culture (Cell Tissue Culture).
Micropropagation The art and science of multiplying plants in vitro. Its implies
- Regeneration- Multiplication
Stages of MicropropagationStage I - Selection & preparation of the mother
plant – sterilization of the plant tissue takes place. Initiation of culture – explant placed into growth media
Stage II – Multiplication – explant transferred to shoot media; shoots can be constantly divided.
Stage III - Rooting– explant transferred to root media
Stage IV - Transfer to soil– explant returned to soil; hardened off
STAGE IV: Transfer to Natural EnvironmentSTAGE III: Pretransplant (rooting)
STAGE II: Shoot ProductionSTAGE I - Sterilization
Steps involved in the in vitro micropropagation
Cleaning of glassware
Preparation of nutrient medium
Selection and sterilization of explant.
Inoculation of aseptic explant into nutrient medium.
Proliferation of shoots on a multiplication medium.
Transfer of shoots for sub-culturing.
Rooting and hardening of plantlets
Field trials.
Cleaning of glassware Graduated measuring cylinders
conical flasks
beakers
Petridishes
pipettes (2 ml, 5 ml and 10 ml)
glass rods
centrifuge tubes
culture tubes
bottles
Procedure for cleaning of glassware
Soak glassware in soap water for 1 hour.
Transfer glassware to conc. HCl and keep for 2 hours.
Rinse glassware in tap water.
Wash the glassware at least twice with distilled water.
Keep glassware for drying in oven at 100 oC for 1 hour.
And then keep glassware in oven at 140-160 oC for 2 hours.
Preparation of nutrient medium
The growth medium used depends upon the plant species to be grown.
The medium contain the following contents:- All of the minerals and vitamins required for the plant
growth and differentiation.- A carbon/energy source such as the sugar as the explant
cannot usually photosynthesize.- Various growth regulators to encourage the cell
elongation, division and differentiation.- Agar which is used to solidify the medium.
Types of medium Chemically defined nutrient medium
Chemically undefined nutrient medium:
Complex additives viz. coconut milk, Casein hydrolysate, yeast
extract, water melon juice, etc. are added in the medium.
1. Solid medium: 6-8% agar-agar
2. Semi solid medium: Less amount of agar
3. Liquid medium: Agar is not added. It is used for cell suspension
culture.
Preparation of stock solutions
It is convenient to prepare stock solutions.
When mixed together in appropriate quantities constitutes
basal medium.
It is not feasible to weigh and mix all the constituents of the
nutrient medium for the preparation of the small quantity of
the nutrient medium.
It also provides flexibility to try different combinations of the
nutrient medium.
Plant Growth regulators
Two major hormones required for the cell differentiation:
i. Auxins - which stimulates root development.ii. Cytokinin - which stimulates shoot
developmentRatio b/w these hormones
i. Auxins Cytokinin = Root developmentii. Auxins Cytokinin = Shoot development
iii. Auxins = Cytokinin = Callus
Selection and sterilization of explant.
Culture medium supports the growth of microbes e.g bacteria, fungi, etc. these grow
fast and kills the plant cells.
Microbes may come from glass vials, instruments, nutrient medium and also from the
plant material.
Therefore, the surface of plant tissue and all non-living articles including nutrient
medium must be sterilized.
Sterilization of non-living articles: The non-living articles viz. Nutrient medium,
glassware, distilled water, instruments (wrapped with brown paper) are sterilized by
autoclaving under steam at a 15 lb/inc2 and temperature 121oC for 15 min. The
glassware can also be sterilized by heating in oven at 150oC for 3-4 hrs. The
thermoilabile compounds are sterilized by passing through the bacterial filters.
Selection and sterilization of explant.
Sterilization of the plant material
(Surface) sterilization The plant material should be surface sterilized to remove the surface
borne micro-organisms.
Water
10% v/v solution of liquid detergent (Teepol) for 10-15 min.
70% ethyl alcohol for 1 min. in front of laminar air flow.
Treatment with 0.1% HgCl2 (W/V) or 5-10% sodium hypochlorite.
Incubation of culture Cultures are incubated in a culture room where light, temperature
and humidity are controlled.
For some tissues dark is essential while for some both dark and
light conditions are required.
Humidity has also some effect.
The cultures are incubated on culture rack at 25-28 oC constant
temperature. Culture tubes are placed at 35-40o inclined position.
Culture to give a light intensity of 4-10 X 103 lux for 16 hrs.
Incubation of culture
Callus FormationCallus is defined as an unorganized tissue
mass growing on solid substrate. Callus forms naturally on plants in
response to wounding, infestations, or at graft unions (Bottino, 1981).
Callus formation is central to many investigative and applied tissue culture procedures.
Effects of culture media, growth regulators and explants on Callus induction and growth: (a-b): Callus induction from nodal explants in Murashige and
Skoog (MS) medium, (c): Callus from leaf explants in MS+coconut water, (d-f): Callus growth in MS medium+2,4 Dichlorophenoxyacetic Acid and Kinetin, (g):
Callus induction in woody plant medium; h and i: Callus growth in MS+α-Naphthalene Acetic Acid and 6-Benzyladenine purine
Subculturing Transfer of cell or tissue from old culture
medium to fresh culture medium within
definite time period.
It provides sufficient space and nutrients to
the growing plantlet.
Multiplication of the callus.
Rooting It is the induction and development of adventitious roots on
the proliferated shoots.
Root formation is induced in a medium with high auxin and
low cytokinins concentrations.
Shoot tip or single node explant is used.
Culture medium is maintained in a green house/mist chamber.
Activated charcoal is frequently added to absorb root-
inhibiting agents.
Rooting
Hardening Healthy/elite plantlets are exposed to the natural conditions in a step wise
manner.
It is a gradual acclimatization of in vitro grown plants to in vivo condition.
The plantlets are transferred to the pots/polyghene bag and immediately
irrigated with inorganic/nutrient solution.
Plants are kept in the hardening room where controlled conditions of light,
humidity and temperature are maintained.
Plants are maintained under high humidity for 10-20 days and subsequently
transferred in the field so as to grow under natural conditions. The success
rate of micropropagation depends on the survival of the plantlets when
transferred from culture to the soil (field).
Hardening
Laboratory setupSpace for washing and storage.
Sterilization room
Inoculation room
Culture room ( incubation room)
Observation and inspection room.
Data collection and management room.
Methods of micropropagationClonal propagation involves the
multiplication of genetically identical lines by asexual reproduction.
Micropropagation involves the use of bud culture, meristem and shoot-tip culture techniques to introduce plants in vitro by induction to form adventitious buds, shoots and entire plants.
Methods of micropropagation
(cont’d..)Bud culture is the culture of plant buds
which contains active meristems.
Meristem culture is the culture of apical meristems which are capable of active cell division and differentiation into specialized and permanent tissue such as shoots and roots
Methods of micropropagation
(cont’d..)Single node culture is done on a hormone
free medium.
Axillary bud culture is done using excised shoot tips cultured on medium amended with high cytokinin concentration 6 Benzyl amino purine(BAP)/Benzyl adenine(BA)
1 2 3
4 5
Surface sterilization in NaOCL
Rinsing with SDW Transfer into sterile container for 2nd sterilization
Steps in single node culture
Methods of micropropagation (cont’d..)
Micro cuttings of yam during and after subculture
3 4
Problems in Micropropagation
There is problem of genetic variability due to Somaclonal variation in some cultures.
Contamination is a major problem which could cause high losses within a short period.
It requires expensive equipments and well trained manpower.
Verification may occur which reduces the rate of growth multiplication of the plant and eventually causes death.
Benefits of micropropagation
Rapid multiplication of superior clones can be carried out through out the year, irrespective of seasonal variations.
Multiplication of disease free plants e.g. virus free plants of sweet potato (Ipomea batatas), cassava (Manihot esculenta) e.t.c.
It is a cost effective process as it requires minimum growing space.