it explains about the plant tissue culture types and stes involved

1. PLANT TISSUE CULTURE
INVITRO
POLLINATION &
FERTILIZATION
A.SHIRLEY
III BIOTECHNOLOGY
BON SECOURS COLLEGE FOR
WOMEN

2. CONTENT
 INTRODUCTION
 HISTORY
 TYPES OF PLANT TISSUE CULTURE
 SELCTION OF EXPLANT
 STEPS INVOLVED IN PLANT TISSUE CULTURE
 INVITRO POLLINATION
 TYPES OF INVITRO POLLINATION
 METHOD OF IN INVITRO POLLINATION
 INVITRO FERTILIZATION
 TECHNIQUES
 APPLICATION S
 CONCLUSION
 REFERENCE

3. INTRODUCTIO
N
TISSUE CULTURE :
 The process of growing cells artificially in the
laboratory is called tissue culture
PLANT TISSUE CULTURE :
 Technique of growing plant cells , tissue and
organs in an artificially prepared nutrient
medium under aseptic condition

4. HISTORY
 1898- Botanist G.Haberlandt
successfully cultured plant
cells isolated from different
tissue
 But 1934-1939 the foundation
 of plant tissue culture was
 laid by three scientists
1. Gauthret
2. White
3. Nobecourt
It was due to the discovery of plant growth
regulators such as auxins and cytokines

5. TYPES OF PLANT TISSUE
CULTURE
TYPES OF
PLANT
TISSUE
CULTURE
Seed
culture Embryo
culture
Meristem
culture
Bud
culture
Callus
culture
Cell
suspensio
n
Anther
culture
Ovary
culture
Protoplas
t culture

6. PLANT TISSUE CULTURE DEPENDS
ON
 TOTIPOTENCY –
It is the ability of a plant cell to regenerate into a
whole plant
 PLASTICITY –
It is the ability of plants to alter their metabolism
growth and development to best suit their
environment
 EXPLANT –
Plant tissue culture are generally initiated from
multicellular tissue obtained from living plants .
Explants may originate from a wide range of plant
tissue such as leaf, stem , roots etc

7. SELECTION OF
EXPLANT : The explant is
selected either
haploid or diploid
 The plant growth
can be achieved in
two ways
1. Shoot directly by
appropriate media
2. Somatic
embryogenesis

8. STEPS INVOLVED IN PLANT
TISSUE CULTURE
1.CULTURE VESSEL :
Erylenmayer flask , conical flask , petri plates and
culture tubes (25 *150mm)
2. CULTURE MEDIUM :
 Murashige and Skoog medium– MS medium
 Gamborg medium – B5 medium
 White medium – W medium
 Nitch medium – N6 medium
They are closed with cotton plug or aluminium foil sheet
PH – 5.8 Acidic

9. 3. STERILIZATION:
To get rid of microbes
Important in plant tissue culture
Autoclave for 121°C 15 mins
Plant tissue should be surface sterilized (dry
heat, flame, autoclave, filter sterilize , wiping with
70% ethanol)
CHEMICAL STERILIZATION :
By treating with any one of chemical like
sodium hypochlorite , calcium hypochlorite, mercury
chloride for 15- 20 mins followed by repeated
washing in sterile water upto 3-5 times

10. .
4. INOCULATION :
Transfer the explant on to a culture medium
called inoculation . It must be done inside the
laminar air flow chamber under aseptic condition .
Flamed and cooled forceps are used of
plant materials to different culture media
5.INCUBATION :
Culture medium with inoculum incubated
26+OR- 2°C with high intensity of light and
allowing photoperiod

11. 6. INDUCTION OF CALLUS :
Due to the activity of auxin and cytokinin
Explant induced Callus
Callus – unorganised mass of undifferentiated tissue
Callus formed by
Auxin -----> cell elongation
Cytokinin ---------> cell division
As a result of which masses of cells are formed
7.MORPHOGENESIS :
Formation of new organs from callus under the influence of
auxin and cytokinin

12. TWO TYPES of MORPHOGENESIS :
i. Organogenesis
ii. Embryogenesis
8. HARDENING:
Exposing the plantlets to the natural environment
in a stepwise is called hardening
Finally plantlets are gradually transferred to the
soil

13. STEPS INVOLVED IN PLANT
TISSUE CULTURE

14. INVITRO POLLINATION
‘Vitro’ means glass or glassy substance . So in vitro
means glass or glass tube .
Cultivation of plant tissue or other organs on artificial
media in a test tube or conical flask is called in vitro
technique
The process of seed formation following sigmatic
pollination of cultured pistil has been referred to as In
vitro pollination

15. TYPES OF IN VITRO
POLLINATION

16. METHOD OF IN VITROPOLLINATION
 The in vitro pollination can be accomplished by
procedure by following appropriate sterilization
procedure, suitable medium, and selection of suitable
explant
 In nature intraspecific hybridization occurs less
frequently . This is due to hindering factor .
 FACTOR – barrier to the growth of the pollen tube on
the stigma or style
 SOLUTION – in such cases the style or part of it can
be excised and pollen grains either placed on the cut
size of ovary
 EXAMPLE – Papaver sommiferum

17. IN VITRO
FERTILIZATION
 The process of seed formation following stigmatic
pollination of cultured pistil has referred to as in vitro
pollination and development of seed through in vitro
fertilization
 DISCOVERY :
 Kranz et al 1990,1991
 Faure et al 1993
 Kranz and Lorz 1993

18. TECHNIQUE
S
SPERMS :
Isolated from pollen grains by osmotic shock in
540 mos mol kg-1 in water mannitol solution
EGG CELL:
Isolated by micro dissection from the ovules
incubated at 40 for 6 minutes
Enzyme solution containing PH 7
 Pectinase (0.75% )
 Pectolyse 1/23 (0.23%)
 Hemicellulose (0.5%)
 Cellulose onozuka RS (0.5%)

19. TECHNIQUE
 The methodology of gametes there in fusion and
culture has been standardised for maize plant
 Isolation of sperm from pollen grains is executed
by osmotic shock in mannitol solution
 Isolation of ovules done by micro dissection of
ovule incubated to 40-60mins
 Micro droplet mannitol solution placed on UV
sterilized cover slip with the aid od micro pillaries
connected to a computated controlled dispenser
to isolate and select the egg cell
 The coverslips the over layed with 300ul
autoclaved mineral oil

20. TECHNIQUE
 By using micro pump isolated sperm is transferred into
micro droplet containing egg
 After fixing, egg is fertilizing by electrofusion because of
supplying maximum of B negative DC pulses
 Fertilize then cultured on a semipermeable transparent
membrane of a ‘Millicell-CM’ dish filled with 0.1 ml of
nutrient solution
 In 50 light intensity 26+or- temperature the dish is then
inserted in the middle of 3cm petriplate where 1.5 of a
feeder cell suspension layer of another maize line remains
 A high frequency of sperm-egg cell fusion was reported
 Fertilized egg divide to form embryos from which
regenerated full plants within 86 days

21. APPLICATIONS
 Micropropagation for ornamental plant increased by
bud proliferation and multiple shoot formation
 Artificial synthetic seeds are produced by somatic
embryos
 Plant tissue culture a plant cell potato and tomato
were brought together there protoplasmic fusion and
hybrid cell was made to develop into a pomato plant
 Useful for mutation breeding , triploidy through
endosperm culture for inducing parthenocarpic fruits

22. CONCLUSION
 Thus plant tissue culture plays a major role in
plant biotechnology
REFERENCE:
Bhojwani S.S and Rajdan M K 1983 – Plant tissue
culture : theory and practice
www.wikipedia.com
www.slideshare.net>mobile
SOURCES:
sciencesamhita.com-tissue culture
pharmatips.in-technique
plantpatch.com-plant
slideshare.net-in vitro pollination types
researchgate.net-invitro fertilization method