Tissue culture is a process that clones plants through micropropagation. It involves culturing plant tissues in sterile conditions with specific nutrients and hormones. There are four main stages - initiation, multiplication, rooting, and acclimatization. The multiplication stage uses cytokinins to induce shoot growth from explants like leaves or stems. Rooting uses auxins to induce root formation from shoots. The process allows for mass production of genetically identical plants independent of seasons.

3. Tissue Culture Technique

4. 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.

5. Haberland (1902) attempted to culture
isolated mesophyll cells but not
succeeded.
Guatheret (1939) cultured callus of carrot.
Miller (1957) put forth the Hormone
hypothesis
S.G. Guha (1966) cultured pollens to
obtain haploid plant.
A.F. Mascarens (1991) induced flowering
in bamboo plant by tissue culture
technique

6. 1) 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.

7. 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.

8. 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.

9.  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.

11. Plant tissue culture(Micropropagation).
Animal tissue culture (Cell Tissue Culture).

13. The art and science of multiplying plants in
vitro.
Its implies
- Regeneration
- Multiplication

14. Stage 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

15. STAGE IV: Transfer to Natural Environment
STAGE III:
Pretransplant (rooting)
STAGE II: Shoot ProductionSTAGE I - Sterilization

16. 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.

17.  Graduated measuring cylinders
 conical flasks
 beakers
 Petridishes
 pipettes (2 ml, 5 ml and 10 ml)
 glass rods
 centrifuge tubes
 culture tubes
 bottles

18. 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.

20. 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.

21.  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.

22.  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.

23.  Two major hormones required for the cell
differentiation:
i. Auxins - which stimulates root
development.
ii. Cytokinin - which stimulates shoot
development
Ratio b/w these hormones
i. Auxins Cytokinin = Root development
ii. Auxins Cytokinin = Shoot development
iii. Auxins = Cytokinin = Callus

24.  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.

26.  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.

27.  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.

29. Callus 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.

31. 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.

34.  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.

36.  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).

38. Space for washing and storage.
Sterilization room
Inoculation room
Culture room ( incubation room)
Observation and inspection room.
Data collection and management room.

39. Clonal 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.

40. 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

41. 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)

42. 1 2 3
4 5

43. 3 4

44. 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.

45. 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.