PLSC 368- Plant Propagation
Lab Exercise 2
January 28, 2009
Name_________________________ Group_________
2. TISSUE CULTURE PROPAGATION
The objectives this lab exercise are to learn how to make plant tissue culture media and get experience in
culturing plants in vitro using several horticultural crops.
A. PREPARATION OF TISSUE CULTURE MEDIA (Jan 28, 2009)
1. Media Composition
Each group will prepare one or two of the following media:
_________________________________________________________________________________
Sucrose
Growth regulators
z
y
No.
Med
(g/l)
(mg/l)
Use
No. of Jars.
_________________________________________________________________________________
1
MS
30
0.2 NAA + 2.0 BA
Shoot multiplication
50
2
MS
30
none
Maintenance
25
3
MS
30
none (liquid medium) Orchid culture
25
________________________________________________________________________________
z
Medium: MS - Murashige and Skoog medium.
y
Growth regulators: naphthaleneacetic acid (NAA), benzyladenine (BA), indoleacetic acid (IAA),
kinetin (KIN).
2. Procedures for Making MS Medium
a. Preparation of inorganic salts.
You may prepare a solution of inorganic salts as shown in the attached handout. In this lab,
however, the Murashige and Skoog (MS) salt mix which has been prepared (Sigma Chemical
Co.) will be used. Dissolve 2.68 g of the salt mix in 500 ml of water contained in a 1-liter flask.
b. Add amino acid and inositol.
Glycine: 2 mg/l
Inositol: 100 mg/l
c. Add vitamins:
Nicotinic acid:
Thiamin HC1:
Pyridoxine HC1:
0.5 mg/l
0.1 mg/1
0.5 mg/l
d. Add sugar or carbohydrate source
Sucrose:
30 g/l, or 20 g/l (see Table)
e. Add growth regulators.
Growth regulator stock solutions have been prepared (most in mg/ml). Add exact amounts of
growth regulators as specified for each medium being prepared.
f.
Bring the medium volume to 1000 ml, using double distilled water and dissolve.
g. Adjust the pH of the solution.
Using a magnetic stirrer, adjust the pH of the solution to 5.7. Use 0.1 N NaOH and 0.1 N HCl
solutions in small quantities. (The pH of the solution must be adjusted before adding agar:
solutions containing agar tend to clog up the electrode of the pH meter).
h. Add agar.
Agar (Difco Bacto-Agar, or Phytogel) 8 g/l
i.
Melt the agar in the solution.
Heat the media on a magnetic stirrer for 30 - 40 minutes until the solution becomes clear. The
media that are to be poured into plates should be autoclaved directly (see step l).
j.
Distribution to culture tubes or jars.
The test tubes are placed in a rack and the medium is distributed into the tubes (20 ml/tube).
k. Put caputs.
Select the color of the caputs according to media being prepared.
l.
Autoclave the media.
Sterilize media by placing the tube racks in the autoclave for 15 minutes at 121oC (240oF) and at
15 PSI. Do not extend heating time. Take the racks out of autoclave as soon as the pressure
drops to zero.
m. Harden the agar medium in slanted tubes.
Transfer the culture tubes to a slanted tube rack while the medium is hot and cool the media in the
slanted tubes.
n. Store media until use.
Place the tube racks in a plastic bag and store them at 4oC until the next week's lab.
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B. CULTURE INITIATION AND SUBCULTURES (Feb 4, 2009)
1. General Procedures for Tissue Culture Propagation
a. Leaf disc cultures (African violet, petunia, salpiglossis, gloxinia, chrysanthemum)
b. Shoot tip cultures (rose, ficus, spirea, birch)
c. Meristem cultures (carnation, potato, banana, syngonium)
d. Runner tip cultures (Boston fern)
e. Protocorm cultures (cattleya, cymbidium)
2. Cultural Procedures
a. Culture initiation (Stage I)
1) Culture of leaf discs
Using a cork borer, take leaf discs (8-mm diam) from young leaves and sterilize them in 5%
Clorox for 10 minutes. Then rinse the leaf discs in sterile water 3-4 times in the transfer hood.
Plant the leaf discs on the agar medium, one disc per tube. Observe for initial swelling and
callus formation from the culture.
2) Culture of shoot tips
Strip off leaves from actively growing branches and obtain shoot buds (apical, axillary),
approximately 0.8-1.2 cm in size. Sterilize them in a 5% Clorox solution for 10 minutes and
rinse them again in sterile water in the transfer hood. Place one explant per tube of solid
medium. Observe shoot growth and proliferation.
b. Subcultures (Stage II)
Divide the shootlets and protocorm masses from Stage II cultures and subculture them for
multiplication. Observe shoot multiplication on your culture; they may be divided continuously
until a large number of plants is obtained. You also will be able to root them as they are large
enough.
c. Rooting of Cultures (Stage III)
Various culture rooting media will be prepared as needed. When your cultures are ready to be
rooted, ask the lab assistant for appropriate media to use. The composition of rooting media
varies with species. Cultures with 2-5 healthy roots can be transferred into soil medium.
d. Plant Establishment in Soil (Stage IV)
A large percentage of plantlets is lost during and after transplanting into soil, mainly due to lack
of plant acclimation. Hence, be careful in handling in vitro grown plantlets at the Stage IV.
Remove agar material carefully from the base of each culture and plant them gently in cell
packs containing peat-lite mixes. Cover the cell pack containers with a plastic sheet for one
week to retain high moisture conditions inside. When plants are well established, you may
transplant them into larger pots.
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Table 1. Chemical compositions of selected plant tissue culture media.
Ingredients
Murashige &
Skoog (MS)
Medium
(1962)
mg/l
Linsmaier &
Skoog (LS)
Medium
(1965)
mg/l
CaCl2
CaCl2 2H2O
Ca(NO3)2 4H2O
CaCl2 6H2O
CuSO4 6H2O
440
Nitsch &
Nitsch
Medium
(1969)
mg/l
White's
Medium
mg/l
160
160
400
300
0.025
0.025
0.025
0.025
0.025
27.8
6.2
27.8
6.2
27.8
10
170
0.83
1,900
370
170
0.83
1,900
370
68
950
185
22.3
22.3
25
Fe2 (SO4)3
FeSO4 7H2O
H3BO3
KCl
KH2PO4
KI
KNO3
MgSO4 7H2O
MnSO4 H2O
MnSO4 4H2O
MoO3
Na2SO4
Na2HPO4
Na2MoO4 2H2O
Na2 EDTA 2H2O
NaH2PO4 H2O
NH4NO3
(NH4)2SO4
ZnSO4 7H2O
Sucrose
Inositol
Folic Acid
Nicotinic Acid
Thiamine HCl
Pyridoxine HCl
Glycine
Biotin
Casain Hydrolysate
IAA
NAA
BA
Agar
pH
0.01 (opt.)
0.25
1.5
65
WPM
Lloyd &
McCown
(1981)
mg/l
Gresshoff &
Doy Medium
(1972)
(modified)
mg/l
96
556
150
0.25
27.8
6.2
0.25
0.25
27.8
3
300
170
0.75
80
720
7
0.001 (opt.)
200
370
0.75
1,000
250
10
29.4
0.25
37.3
30.0
0.25
37.3
90
0.25
37.3
0.25
37.3
0.25
37.25
1,650
1,650
720
8.6
8.6
10
3
30,000
100
30,000
100
20,000
100
0.5
5
0.5
0.5
2.0
0.05
20,000
20,000
10
0.5
0.1
0.1
3
0.1
1.0
0.1
16.5
0.5
0.1
0.5
2.0
0.4
400
8.6
200
3
10 (opt.)
0.1
10,000
5.7
10,000
8,000
5
5,000
2.0
5.0
7,000
5.7-5.8
PLSC 368 - Plant Propagation
Lab Exercise 2: Questions
Spring Semester, 2009
Name__________________________ Group_________
2. TISSUE CULTURE LAB REPORT
(Due April 22, 2009)
1. List the all ingredients of Murashige and Skoog (MS) medium in ppm concentrations.
2. Show the concentrations of N, P, K, and Ca in the MS medium used:
Chemical forms
ppm
mM
meq/l
+
Ammonium (NH4 )
________
________
________
Nitrate (NO3-)
________
________
________
Total elemental N
________
________
Total elemental P
________
________
________
Total H2PO4________
________
________
Total K+
________
________
________
Total calcium (Ca2+)
________
________
________
_______________________________________________________________________
Calculations:
3. Calculate the concentrations of the following growth regulators:
Growth Molecular
regulator weight
NAA
IAA
KIN
BA
186.2
175.2
215.2
225.3
0.1 M
________
________
________
________
Amount/liter (g, mg, or µg per liter)
1 M
10 mM
1 mM
µM
________
________
________
________
________
________
________
________
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_________
_________
_________
_________
________
________
________
________
4. Why an agar medium containing growth regulators can not be over-sterilized in an autoclave?
5. Discuss the advantages of shoot tip cultures over leaf disc culture in propagation.
6. Discuss advantages of protocorm culture over seed propagation in orchid production.
7. Discuss problems that you have encountered in each stage of culture:
a. Stage I cultures:
b. Stage II cultures:
c. Stage III cultures:
d. Stage IV cultures:
8. Briefly describe what you have accomplished in this lab.
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