Tissue culture....regards manjunath

1. BY:
MANJUNATHA G A
2013-11-176
Dept. of PBGN

2. PlantTissue culture
It is a technique of growing cells, tissues, organs or whole
organism in vitro (in glass) on artificial culture medium under
aseptic and controlled conditions.
Haberlandt (1854-1945) attempted to cultivate plant tissue
culture cells in vitro. He is regarded as the father of plant tissue
culture.

3. Plant Tissue Culture: historical highlights
1902: Haberlandt attempted to the culture mesophyll tissue and root hair
cells.
This was the first attempt of in vitro culture.
1904: Haning attempted to culture excised embryos from mature seeds.
1922: Kotte was successful in obtaining growth from isolated root tips on
inorganic media. Robbins reported similar success from root tip and
stem tip.
1934: Used yeast extract (vit B) with inorganic salts to repeatedly culture
root tips of tomato.
1935: Importance of B vitamins and PGRs in culture of mesophyll cells.
1939: Tobacco crown gall culture, callus obtained: called as Plant Cancer.
1940: Coconut milk used in plant cultures to obtain heart-shaped embryos.
1950s:Skoog used adenine sulfate to obtain buds on tobacco segments:
1958: Stewart and Reinert obtained somatic embryos from carrot cells
using PGRs.
1950-60s: Botanists turned to plant tissue culture to study plant
development.

4. 1960: Cocking isolated protoplasts from cultured cells.
1962: Murashige and Skoog developed MS media for
tobacco.
1966: Guha and Maheshwari obtained first haploid plants
1970: Discovery of restriction endonuclease (Daniell
Nathan).
1972-73: First recombinant molecule created by Stanley
Cohen.
1974: Discovery of Ti plasmid in Agrobacterium
tumefaciens (by Zaenen)
1983: First transgenic plant. (Monsanto).
1985: Leaf disk transformation method (Monsanto)

5. •Culture:- Growing of cells, tissues, plant organs (or)
whole plants in nutrient medium under aseptic
conditions.
•Explant : An organ, tissue or a cell obtained from a
donor parent and used for culture in vitro.
Depending upon the explant source it can be named as
follows :
•Anther : anther culture
•Pollen : pollen culture
•Embryo : Embryo culture
•Cell : Cell culture
•Protoplast : Protoplast culture
•Callus : callus culture

6. Callus Culture
Cell-suspension Culture
Tissue or Organ Culture
• Shoot tip/ apical meristem culture
• Axillary Bud culture
• Root culture
• Ovary culture
• Embryo culture
• Anther culture
Protoplast Culture
Culture Types

7. Callus:
An amorphous mass of loosely arranged
thin-walled parenchyma cells arising from
the proliferating cells of the parent tissue
cultured on agar medium.

8. PRODUCTION OF CALLUS FROM
EXPLANT
•Sterilized explant is transferred aseptically onto defined
medium.
•Transfer to BOD incubator.
•Temperature (25 ± 2 ͦ) and light is necessary for callus
production.
•Callus produced with in 3-8 days.

9. Suspension culture
•When friable callus is placed into a liquid medium
(usually the same composition as the solid medium
used for callus culture) and then agitated single cells
and / or small clumps of few to many cells are
produced in the medium is called suspension culture.
•Liquid cultures may be constantly agitated generally
by a gyratory shaker of 100-250 rpm to facilitate
aeration and dissociation of cell clumps into small
pieces.
•Suspension cultures grow much faster than callus
cultures, need to be sub-cultured at every week, allow
a more accurate determination of the nutritional
requirement of cells and even somatic embryos.

10. The suspension culture broadly grouped as
1) Batch culture 2) Continuous culture
•1) Batch culture :
A batch culture is a cell suspension culture
grown in a fixed volume of nutrient culture
medium.
Cell suspension increases in biomass by cell
division and cell growth until a factor in the
culture environment (nutrient or oxygen
availability) becomes limiting and the growth
ceases.

11. 2) Continuous culture:-
These cultures are maintained in a steady state for
a long period by draining out the used (or) spent medium
and adding the fresh medium.
such subculture systems are either closed (or) open type.
1) Closed:- The cells separated from used medium taken
out for replacement and added back to the suspension
culture, So that the cell biomass keeps on increasing.
2) Open:- Both cells and the used medium are takenout
from open continuously cultures and replaced by equal
volume of fresh medium.The replacement volume is so
adjusted that cultures remain at sub-maximal growth
indefinitely.

12. Organ culture:
•Culture of isolated plant organs such
as root tips, shoot tips, leaf
primordial, immature parts of flower
etc.

13. Meristem culture
 Cultivation of auxiliary or apical shoot meristems,
particularly of shoot apical meristem ,is known as
meristem culture.
Applications:
Production of virus free
germplasm.
 Mass production of desirable
genotypes.

14. Root culture

15. Ovary/ovule culture:
Ovary or ovule culture involves development of
haploid from unfertilized cells of embryosac
present in ovary.
Applications:
*Production of haploid plants.
*Achievement of in vitro fertilization.

16. SOMATIC EMBRYOGENESIS:
In somatic embryogenesis the embryos regenerate
from somatic cells, tissue or organs either directly from
the tissues or from callus of explant.
Direct embryogenesis
•The embryos initiate directly from explants in the absence of
callus formation.
Indirect embryogenesis
•Callus from explants takes place from which embryos are
developed

17. Production of artificial seeds.
As source materials for embryogenic protoplast.
For genetic transformation.
Production of primary metabolites specific to seeds
such as lipids in oil seeds

18. ANTHER CULTURE/POLLEN CULTURE:
Anther culture is a technique by which the
developing anthers from unopened flower bud are
cultured on a nutrient medium where the
microspores within the cultured anther develop
into callus tissue or embryoids that give rise to
haploid plant.
Applications:
Production of haploid plant
Production of useful gametoclonal varition.

19. PROTOPLAST CULTURES:
Isolated protoplasts have been described as "naked"
cells because the cell wall has been removed by either a
mechanical or an enzymatic process.
Protoplasts can be induced to reform a cell and divide if
placed in a suitable nutrient medium than form callus.

20. Cloning:
•Clone:
Group of cells obtained from a single cell
through mitosis, which have same genotype so
that any variation among them is expected to be
only environmental.
•Cloning:
process of obtaining clones; in case of cells
or in case of organisms etc.

21. The Steps in the Plant Cloning Process are:
•Small amounts of tissues or cells are taken
from a plant.
•The cells are then transferred to plates
filled with sterile nutrient agar jelly.
•Auxins are then added to prepare all
the cells for the process of mitosis.
•Small masses of tissue grow at a fast rate.
•Growth hormones are then added to
prepare the long process of root and stem
growth.
•The tiny plantlets are then put into potting
trays where they develop into adult plant.

22. Advantages of Plant Cloning
• Duplication can lead to more plants in the world
• A continuous supply of plants forever (some cloned plants are still alive
from hundreds of years ago
• The cloning procedure (grafting) can make genetically different root and
shoot systems to make a plant more better, stronger, longer lasting and
have better qualities than the original
• Plants can be made to not be infected by diseases and viruses
• The Plant Cloning Process is sometimes less expensive than crop growing
• The pollination of plants may not be needed on cloned plants
• Some seeds are difficult to obtain or germinate so cloning is a preferable
alternative.
• Much more profit to the food industry and the plant industry.

23. Regeneration
• In tissue culture; development of organized structures like root,
shoot, somatic embryo etc. from callus/cell culture.

24. Steps in plant tissue culture technique
•Selection of plant
•Isolation of explant
•Sterilization of explant
•Inoculation of explant

25. •Incubation
•Initiation of callus
•Sub culturing
•Regenaration
•Hardening
•Transfer of plantlets to Green house or open
field

26. Applications of plant tissue culture in crop improvement
• 1. Micro propagation helps in mass multiplication of plants which are
difficult to propagate through conventional methods.
• 2. Some perennial crop plants like ornamental and fruit crops can not be
propagated through seeds.The vegetative propagation like grafting,
budding are tedious and time consuming. In such crops micro
propagation helps in rapid multiplication.
• 3. Rapid multiplication of rare and elite genotypes such asAromatic and
Medicinal plants.
• 4. Screening of large number of cells in small space.
• 5. Cross pollinated crops like cordamum, Eucalyptus, coconut, oil palm
do not give true to type plants, when multiplied through seed.
Development of genetically uniform plants in cross pollinated crops is
possible through tissue culture .

27. •6. In case of certain horticultural crops orchids etc seed will not
germinate under natural conditions, such seed can be made to
germinate in vitro by providing suitable environment.
• 7. Induction of flowering in some trees that do not flower or delay
in flowering. Eg:- Bamboo flowers only once in its life time of 50
years
• 8.Virus free plants can be produced through meristem culture
•9. Large amount of germplasm can be stored within a small space
and lesser cost for prolonged periods under in vitro condition at
low temperature.The preservation of cells tissues, organs in
liquid Nitrogen at – 196OC is called cryopreservation
• 10. Production of secondary metabolites. Eg:- Caffine from
coffea arabica, Nicotine from Nicotiana rustica.
• 11. Plant tissue culture can also be used for studying the
biochemical pathways and gene regulation.

28. •12. Anther and pollen culture can be used for production of
halploids and by doubling the chromosome number of haploids
using cholchicine homogygous diploids can be produced.They
are called dihaploids.
•13. In case of certain fruit crops and vegetative propagated plants
where seed is not of much economic important, triploids can be
produced through endosperm culture.
•14. Inter specific and inter generic hybrids can be produced
through embryo rescue technique which is not possible through
conventional method. In such crosses in vitro fertilization helps
to overcome pre-fertilization barrier while the embryo rescue
technique helps to over come post fertilization barrier.
•15. Somatic hybrids and cybrids can be produced through
protoplast fusion (or) somatic hybridization
•16. Ovary culture is helpful to know the physiology of fruit
development.
•17. Development of transgenic plants etc..