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Tissue culture, nutrient stock solutions and

media preparation for in-vitro cultures


R. Muthuraj & Sumita
Senior Scientist, Division of Seed Technology,
Central Potato Research Institute, Shimla-171001.
Now a days all major tissue culture media formulations are available in
powdered from. In spite of their wide use and comparative ease in handling, storage
and media preparation, they are expensive. Besides, the use of powered media and
minor elements. Therefore, the classical approach of nutrient stock preparation and
their proportionate mixing to obtain a desired volume of the culture medium at a
prescribed concentration is still practiced in all the laboratories across the world. This
approach is not only convenient to prepare different media using common stock as and
solutions, but also provides a scope for periodical replenishment of nutrient stocks as
and when required .For plant tissue culture media preparation, mainly five different
stock solutions are required. A general protocol for the preparation of nutrient based on
MS medium is given below. Nutrient stock solutions of other media formulations can
well be prepared following the same guidelines.
NUTRIENT STOCK SOLUTIONS BASED ON MS MEDIUM
I.

Macro stock (MS-I) in 1000 ml.


S.No. Chemicals
1.
2.
3.
4.

KNO3
NH4NO3
KH2PO4
MgSO4.7H2O
Vol.ml per liter

10x
19.0
16.5
1.7
3.7
100.0

Quantity in gm.
20x
40x
38.0
76.0
33.0
66.0
3.4
6.8
7.4
14.8
50.0
25.0

100x
190.0
165.0
17.0
37.0
10.0

Take 500 ml double distilled water in a 2.0 liter beaker, weight, add and keep on
dissolving each salt sequentially in a descending order (dissolve by magnetic stirring)
and finally make up the volume to 1000 ml by double distilled water. Store at 4 C.
II. Calcium stock (MS-II) in 1000 ml.
S.No.
1.

Chemicals
CaCl2 .2H2O
Vol.ml per liter

10x
4.4
100.0

Quantity in gm.
20x
40x
8.8
17.6
50.0
25.0

100x
44.0
10.0

Take 500 ml double distilled water in a 2.0 liter beaker, weight, add CaCl2 .2H2O and
keep on dissolving (dissolve by magnetic stirring) and finally make up the volume to
1000 ml by double distilled water. Store at 4 C.

III Micro stock (MS-III) in 1000 ml.


S.No.

Chemicals

1.
2.
3.
4.
5.
6.
7.

H3BO3
MnSO4. H2O
ZNSO4.7H2O
KI
Na2MoO4.2H2O*
CuSO4.5H2O**
Cocl2.6H2O**
Vol.ml per liter

10x
62
168.9
86
8.3
2.5
0.25
0.25
100.0

Quantity in gm.
20x
40x
124
248
337.8
675.6
172
344
16.6
33.2
5
10
0.5
1
0.5
1
50.0
25.0

100x
620
1689
860
83
25
2.5
2.5
10.0

*Prepare separately and then add.


**Prepare 100 mg in 100 ml DDH2O and then add required quantity (for 1000 ml
of 100X-25 ml)
Take 500 ml double distilled water in a 2.0 liter beaker, weight, add and keep on
dissolving each salt sequentially in a descending order (dissolve by magnetic stirring)
and finally make up the volume to 1000 ml by double distilled water. Store at 4 C.
Note that Na2MoO4. 2H2O should be dissolved separately in approximately 200 ml of
double distilled water and added to the stock after dissolving ZnSO 4. 7H2O. Also note
that MnSO4. H2O takes time to dissolve.
IV. MS Iron EDTA stock (MS-IV) in 1000 ml
S.No.

Chemicals
10x

1.
2.

Quantity in gm.
20x
40x

100x

Na2
EDTA.
2H2O
FeSO4. 7H2O
Vol.ml per liter

Take 1000 ml double distilled water in 1500 ml amber coloured bottle and warm the
water near boiling. Now weight and add Na2 EDTA. 2H2O while stirring under
magnetic stirrer; after Na2 EDTA. 2H2O has been dissolved add gradually FeSO4.
7H2O under mild magnetic stirring. Immediately after adding FeSO 4. 7H2Oclose the
bottle and keep on stirring at least for an hour. store at 4 C.
V. Vitamin stock (MS-V) in 1000 ml.
S.No.
1.
2.
3.
4.
5.

Chemicals
Myo- inositol
Glycine
Thiamine HCL
Nicotinic acid
Pyridoxine HCI
Vol.ml per liter

10x
1000
20
1
5
5
100.0

Quantity in gm.
20x
40x
2000
4000
40
80
2
4
10
20
10
20
50.0
25.0

100x
10000
200
10
50
50
10.0

Take 500 ml double distilled water in a 2.0 liter beaker, weight, add and keep on
dissolving each salt sequentially in a descending order (dissolve by magnetic stirring)
and finally make up the volume to 1000 ml by double distilled water. Store at 4 C.
Vitamin stock is very prone to microbial contamination. Therefore always check the
stock before use.
VI. Growth regulator stocks: Always prepare growth regulator (auxins, cytokinins,
gibberellins, ets.) stocks at a concentration of 1.0 mg / ml. However, if the growth
regulator concentrations ( in the media) are expressed in micromoles, it is
recommended to prepare their stock solution in millimolar or micromolar strength.
This would help avoid unnecessary lengthy calculation during medium preparation.
MEDIA PREPARATION: Mixing of stock solutions:
For the preparation of MS medium, the above stock solutions should be added
sequentially. The quantity of stock should be added for preparing different quantities
of media depends on the concentration of stocks prepared. The following table gives
an idea about quantity of stocks to be used for preparing different quantity of media.
S.No. Stock
Quantity in ml or gm per litre.
solution
1
2
3
4
5
6
1.
MS-I (40X)
25.0
50.0
75.0
100.0
125.0
150.0
2.
MS-II (40X)
25.0
50.0
75.0
100.0
125.0
150.0
3.
MS-III (40X)
25.0
50.0
75.0
100.0
125.0
150.0
4.
MS-IV (40X)
25.0
50.0
75.0
100.0
125.0
150.0
5.
MS-V (40X)
25.0
50.0
75.0
100.0
125.0
150.0
6.
Sucrose (gm)
30.0
60.0
90.0
120.0
150.0
180.0
7.
Agar (gm) or
8.0
16.0
24.0
32.0
40.0
48.0
8.
Gelrite (gm)
2.0
4.0
6.0
8.0
10.0
12.0
For growth regulator the stock solution should be prepared 1st by dissolving it in
appropriate solvents. For the weight 100mg of growth regulator and dissolve it with
small quantity of solvent and make the volume to 100 ml with double distilled water for
further use (100mg in 100 ml).
9.
GA3
0.10
0.20
0.30
0.40
0.50
0.60
10.
NAA
0.01
0.02
0.03
0.04
0.05
0.06
11.
Calcium
2.00
4.00
6.00
8.00
10.00
12.00
dpentatinate

The method is exemplified for preparing 1.0 liter of MS basal medium.


a. For preparation of 1.0 liter of MS Basal medium, the above stocks solutions
should be added sequentially in about 500 ml of doubled distilled water.
b. Weight and add required quantities of sucrose (20 or 30 g ) and dissolve by
magnetic stirring. According to the purpose of the medium growth regulator and
other medium conjugates/ additives are added, and the volume of the medium is
made up to 1000 ml by distilled water. Note that thermo-labile growth regulators
and additives should be added by filter sterilization only after autoclaving.

c. Adjust the of the medium to 5.8 using 0.1 NaOH or 0.1 N HCL before autoclaving.
Note that the meter should be calibrated by standard buffers (
4.0 and 7.0)
immediately before adjusting the medium.
d. For preparing semisolid medium, add agar at the rate of 6.0-8.0 grl and gelrite at
the rate of 2.0 g/l in the pH adjusting medium, and heat until near boiling in a
microwave oven or gas oven with intermittent stirring. Measured volume of
semisolid media is dispenser. Into culture tubes, containers. Vessel using a
automatic media disperser. For preparing liquid medium, pH adjusted media are
directly poured in suitable containers .For plating experiments, semisolid medium
is poured in sterile petri dishes under a LFCA workstation.
e. Plant tissue culture media are usually autoclaved at 121 C For 20 min (15 lb in or
1.05 kg cm2). Autoclaving is generally done in a horizental or vertical autoclave.
Minimum time necessary for steam sterilization of media is dependent on volume
of medium per vessel and is described separately.
f. Autoclaved media are kept in ambient temperature for a day and then transferred
in a dust-free closed cabinet for subsequent use. Semisolid medium starts drying
up, and therefore should be used within a fortnight after its preparation.
Minimum autoclaving time for plant tissue culture medium
S.No.
Medium volume/ vessel (ml)
Minimum autoclaving time (min)
1.
25
20
2.
50
25
3.
100
28
4.
250
31
5.
500
35
6.
1000
40
7.
2000
48
8.
4000
63
Burger, 1988