:

History and development of tissue culture

.


.
.
)) Schwann,1839
.

.
) (Haberlandt, 1902

.

.

-:

(
(1902
) (.
(Hannig, 1904
.
(1934
.
(White, 1934
.
(Nobecourt, 1937

18

Haberlandt,

)
)Gautheret,

)
)

) ( .
)(Gautheret and Nobecourt, 1939

 .
(White, 1939 .
(

.
)(Skoog, 1944

)Skoog
.
(and Tsui, 1948
)(Ball, 1950
.

.
)(Morel and Martin, 1952
Muir et al.,
.
) (1954
.
Miller et

(al.,1955
)
)(Tulecke and Nickell, 1956
(.

)Skoog and
) (.
(Miller, 1957
)Morel,
.
(1960
) (MS
)Murashige and
.
(Skoog, 1962
)Power et al.,
.
(1970
.
)(Takebe et al., 1970
) (Biotransformation
)
.
(Reinhard, 1974

19

 ) (Ti-plasmid

(Zaenen et al., 1974
.
(
) (Ti-plasmid DNA
)Chilton et al.,
.
(1977
.
)(Larkin and Scowcroft, 1981
)
.
(Zimmermann, 1982
.
)(Paszkowski et al.,1984

)Horsch et
.
(al., 1985
PCR
Mullis 1993
.

20

 :

)
(............
.
Micropropagation .

) .
(


.

-1
) (

.

.
-2

)(
.
-3
Explant : 5-3

21


.
-4

)(

.
-5

.

-6
.
-7 .
)

(

:
-1
.
-2
.UV
-3 .

22

 -4
.
-:
-1

-2

.
-3

.
-4

23

 Tissue culture :

.



8.6 %58
.

.
.
:
-1

.
-2

.

.
-3

Somaclonal Variation

.
-4

24

-5

) (

.
:
:


.
:


.
:



.
:

-1
.

25

-2

-3

10 . %20

-4

.
-5

.
:
:
)(


.
:

(Bt corn) . " "
" : " .

:
-1 41% 27 % .
-2 25 % .
-3
STRIVE.

26

Explants can be pieces of any part of the plant (leaves, stems, flowers, etc. (,
or even individual isolated cells.

The tissue will begin to grow. It may make a big blob of tissue called callus, or it may
make new shoots directly from the explant tissue that was inserted in the container.

27

A mass of callus tissue is formed that is just starting to make new plantlets.

New plantlets (shoots with leaves) are forming.

28

If the conditions are right a small "forest" of plants will develop in the tissue culture container
Some of the small plantlets can be removed and transferred to new tissue culture
containers.
These will produce more shoots and fill the container.

29

When the plantlets are large enough, they can be removed from the tissue culture container and
transferred into pots with potting soil. The young plants are growth in a greenhouse just like
you would any young seedling or cutting When the small plant clones are removed from the
culture containers, they must be transplanted into some type of acclimation container or kept
under a mist system until the acclimate to the ambient environment.

30

After acclimation, the young plants can be transplanted

and grown in pots in a greenhouse to produce new plants.

31

 -:
4 :
- 1 Kitchen area :

) (
.D.D.W .
- 2 Cultivation Room :

.
- 3 Growth Room :

-: 25 16
.
- 4 Mist Room :

.
.

.
:
:Rapid clone Propagation - 1
.
-: True - to - type -

32

 -
.
" " Clone -:
.
-: Rapid -

6 -4 .
-: Mass production -

.
)(Germplasm Storage and exchange -:

-



.
-



.
-:
Production of pathogens [mainly virus] - free plants

-: - - -.
0.5 - 0.2 .

33

 Vessels
.

.
Pharmacology, Vaccines -:

-:
- - - -.

Callus
. -:
Vaccines .

Mushroom Tissue Culture

34

35

The father of plant tissue culture is considered to be the German Botanist HABERLANDT
who conceived the concept of cell culture in 1902.

36

37

 Establishment of tissue culture lab :

:
-1 .
-2 .
-3 .
.
.
:
:
-1 Laminar flow air .

-2 .
-3 - - .
-4 1,5 -1 .
-5 ) % 5,5 ( -
.Tween-20
-6
-7 Centrifuge
Protoplast isolation

-8
: :
-1 .
-2 Incubators

38

 .
-3 Shaker Cell suspension cultures
.Embryo cultures
-4 .
-5 .
:
.
: -:

:
) (. )(. ) (C12H22O11 30 . ) ( 10-6 ) ( .Liquid medium
:
- :
:
-1 ) :(N
Nuclic acids .Amino acids
) (NH4+ ).NO3)-

39

 -2 ) :(P
. ((KH2PO4
). (NaH2 PO4
-3 ) :(K .
KNO3 . KH2PO4
.KCl
-4 ) :(S . .
).( =SO4
-5 ) :(Ca
. CaCl2
.Ca(NO3)2
-6

) :(Mg . .

. .MgSO4
-7

) :(Fe .

.
FeSO4 ethylene-diamine tetraacetic

(acid (EDTA .
- :
.
-1 Boron ) (B Boric acid

.H3BO3
-2 Molybdenum ) (Mo
Sodium molybdate .Na2MoO4.2H2O

40

 -3 Manganese .MnSO4
-4 Co 25
.CoCl2
-5 Zn ZnSO4.
-6 Cu 25 CuSO4

.5H2O .
-7 Cl CaCl2 .
-8 Iodine ).(KI
: :
(Skoog& Murashige (MS, 1962

Woody Plant Medium ((WPM

:
:

41

.Culture of Meristem-tip

.Culture of Shoot-tip

.bad) Culture of Axillary (lateral

.Single node culture

42

:
:
:
:
:
MS) Skoog, 1962 & Murashige):
1

Macronutrients:
Ammonium nitrate) NH4NO3) 1,650 mg/l
Boric acid (H3BO3) 6.2 mg/l
Calcium chloride (CaCl2 H2O) 440 mg/l
Cobalt chloride) CoCl2 6H2O) 0.025 mg/l
Magnesium sulfate) MgSO4 7H2O) 370 mg/l
Cupric sulfate) CuSO4 5H2O) 0.025 mg/l
Potassium phosphate) KH2PO4) 170 mg/l
Ferrous sulfate) FeSO4 7H2O) 27.8 mg/l
Potassium nitrate) KNO3) 1,900 mg/l
Manganese sulfate (MnSO4 4H2O) 22.3 mg/l
Potassium iodine (KI) 0.83 mg/l
Sodium molybdate) Na2MoO4 2H2O) 0.25 mg/l
Zinc sulfate (ZnSO 47 H2O) 8.6 mg/l
Na2EDTA 2H2Oa 37.2 mg/lb

Common organic additives:

i-Inositol 100mg/l
Niacin 0.5 mg/l
Pyridoxine HCl 0.5 mg/l
Thiamine HCl 0.1 mg/l
IAA 1-30mg/l
Kinetin 0.4-10 mg/l
Glycine (recrystallized) 2.0 g/l
Edamine 1.00 g/l
Sucrose 20 g/l
Agar 10 g/l

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