Modified Soybean Transformation Protocol for the Cot-Node Method
Paula Olhoft
1991 Upper Buford Circle, 411 Borlaug Hall
University of Minnesota, Dept. of Agronomy and Plant Genetics
St. Paul, Minnesota 55113
olhof003@tc.umn.edu
612-625-8107
Seedling preparation
A. Soybean seeds are gas sterilized 24 hours in a chamber with a tightly fitting lid.
-
One layer of seeds are placed in a 15 x 100 mm plastic Petri dish
Add 100 ml chlorox to a 250 ml beaker
Slowly add 3.5 ml 12N HCl
Cover completely
A longer gas sterilization time may be required for those beans with bad mold/fungus
problems.
B. Sterile soybean seeds (16) are placed on Germination Medium (GM) and grown 5-6 days or
until the cotyledons turned green but before the first leaves grow out of the cotyledon.
Germination Medium:
B5 Major Salts (10x)
B5 Minor Salts (100x)
MSIII Iron Stock (100x)
Sucrose (2%)
B5 Vitamins (100x)
0.8% Purified Agar BBL
pH 5.8
100 ml/l
10 ml/l
10 ml/l
20 g/l
10 ml/l
8 g/l
Agrobacterium Preparation
A. The Agrobacterium (from a stock tube) is first streaked onto a solid Agrobacterium growth
medium and incubated for about 2 days at 250C or until colonies appear. Unless a
minimum nutrient medium is required (eg. AB medium), YEP is used for both solid and
liquid medium.
YEP Medium:
Bacto-peptone
10 g/l
Yeast-extract
5 g/l
NaCl
5 g/l
Difco granulated agar
12 g/l
PH 7.0
+ Appropriate antibiotics for selection
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B. A single colony is picked with a toothpick and placed in 50 ml of liquid Agrobacterium
growth medium and shaken on a rotating table at 250C (175 rpms). After an OD260
between 0.8-1.0 is reached (usually 2 days), a 15% glycerol stock is made with the broth
and 1-ml aliquots are made and stored at –800C.
C. The day before explant inoculation, 3ml of the glycerol stocks plus appropriate antibiotics are
added to 200-ml YEP liquid medium in a 500 ml Erlenmeyer flask. The flask is shaken
at 250C until an OD260 between 0.8-1.0 is reached.
D. Before cutting and inoculating the soybean explants, the broth is divided into 50 ml aliquots
and the Agrobacterium pelleted in 50 ml Falcon tubes by centrifuging for 10 min at
3,270 x g at 200C. The pellet is then resuspended in 25-ml liquid co-cultivation medium
and allowed to sit at room temperature for 30 minutes before use.
Co-Cultivation Medium:
1/10th B5 Major Salts (10x)
1/10th B5 Minor Salts (100x)
1/10th MSIII Iron Stock (100x)
Sucrose (3%)
2-[N-morpholino]ethanesulfonic
acid (MES) 20mM
0.5% Purified Agar BBL (solid)
pH 5.4
*B5 Vitamins (100x)
*Acetosyringone (AS) 200 μM
*Gibberellic acid (GA) 0.7 μM
*6-benzyl-aminopurine (BAP) 7.5 μM
**L-Cysteine 3.3 mM or 8 mM
**Dithiothrietol (DTT) 1 mM
10 ml/l
1 ml/l
1 ml/l
30 g/l
3.9 g/l
5 g/l
10 ml/l
40 mg/l
0.25 mg/l
1.67 mg/l
400 mg/l or 1 g/l
154.2 mg/l
* Components are mixed together and filter-sterilized (Acrodisc 25 mm Syringe
filter with 0.2 μM membrane) into cooled, autoclaved medium.
** Components are varied and can be combined with one another. These are
separately filter-sterilized and added to the co-cultivation medium as well.
Explant Preparation
A. For each seedling, the roots and the majority of the hypocotyl are removed approximately 3-5
mm below the cotyledonary node by cutting with a number 15 Personna Plus surgeon’s
blade (American Safety Razor Company, Staunton, Virginia). Two explants are obtained
by separating the cotyledons and cutting vertically through the hypocotyl region. The
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epicotyl is removed and the axillary bud and the cotyledonary node are wounded by
cutting 10 times with the blade perpendicular to the hypocotyl.
B. The explants are incubated in the 25-ml co-cultivation/Agrobacterium suspension for 30
minutes before randomly placing (adaxial side down) five explants on sterile Whatman
#1 (70 mm) filter paper (Whatman International Ltd, Maidstone, England) on top of solid
co-cultivation medium. Five plates are then wrapped with Parafilm “M”(American
National Can, Chicago, Illinois) and incubated at 250C for 5 days in the dark.
Selection and Plant Regeneration
A. After five days incubation, the explants are washed in a liquid shoot induction medium to
remove excess Agrobacterium and subsequently imbedded into a solid shoot induction
medium without hygromycin, five per plate. Plates are wrapped with Scotch 394 venting
tape (3M, St. Paul, Minnesota) and placed in a growth chamber for two weeks with a
temperature averaging 250C under 18 h light/6 h dark cycle at 90-150 μE/m2s.
Shoot Induction Medium (SI):
B5 Major Salts (10x)
B5 Minor Salts (100x)
MSIII Iron Stock (100x)
Sucrose (3%)
2-[N-morpholino]ethanesulfonic
acid (MES) 3 mM
0.8% Purified Agar BBL
pH 5.6
*B5 Vitamins (100x)
*6-benzyl-aminopurine (BAP) 7.5 μM
*Ticarcillin (TICAR)
*Cefotaxime (Claforan)
100 ml/l
10 ml/l
10 ml/l
30 g/l
0.59 g/l
8 g/l
10 ml/l
1.67 mg/l
500 mg/l
100 mg/l
* Components are mixed together and filter-sterilized (Acrodisc 25 mm Syringe
filter with 0.2 μM membrane) into cooled, autoclaved medium.
B. After two weeks, the explants are sub-cultured into new shoot induction medium with 5 mg/l
hygromycin* after carefully removing the hypocotyl and placed back into the growth
chamber for an additional 14 days.
* Only if hygromycin is your selective agent (stock is 50 mg/ml)
C. After two weeks, the explants are sub-cultured into shoot elongation medium with 10 mg/l
hygromycin after removing the cotyledon and placed back into the growth chamber for
14 days.
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Shoot Elongation Medium (SE):
MS Major Salts (10x)
MS Minor Salts (100x)
MSIII Iron Stock (100x)
Sucrose (3%)
2-[N-morpholino]ethanesulfonic
acid (MES) 3 mM
0.8% Purified Agar BBL
pH 5.6
*B5 Vitamins (100x)
*L-Asparagine
*L-Pyroglutamic acid
*Indole-3-acetic acid (IAA)
*Gibberellic acid (GA)
*Zeatin-riboside
*Ticarcillin (TICAR)
*Cefotaxime (Claforan)
*Hygromycin
100 ml/l
10 ml/l
10 ml/l
30 g/l
0.59 g/l
8 g/l
10 ml/l
50 mg/l
100 mg/l
0.1 mg/l
0.5 mg/l
1 mg/l
500 mg/l
100 mg/l
10 mg/l
* Components are mixed together and filter-sterilized (Acrodisc 25 mm Syringe
filter with 0.2 μM membrane) into cooled, autoclaved medium.
D. Explants need to be sub-cultured every 2-3 weeks depending on how effective selection is at
the moment. The level of selection, hygromycin, will need to be adjusted between 0-10
mg/l depending on the response of the explant. AIM: Get a nice healthy transgenic
shoot mass growing vigorously before the entire explant dies.
E. After a healthy, vigorous shoot has at least three sets of leaves and preferably over 4 cm in
length, place in rooting medium after excising at an inter-nodal stem position. Roots
form after 5 days and usually up to 2 weeks.
Rooting Medium:
½ B5 Major Salts (10x)
½ B5 Minor Salts (100x)
MSIII Iron Stock (100x)
Sucrose (2%)
2-[N-morpholino]ethanesulfonic
acid (MES) 3 mM
0.8% Purified Agar BBL
pH 5.6
*Indole-3-butyric acid (IBA)
50 ml/l
5 ml/l
10 ml/l
20 g/l
0.59 g/l
8 g/l
1 mg/l
* Component must be filter-sterilized and added to cooled, autoclaved medium
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F. Rooted shoots can be directly transferred to soil and placed in a growth chamber or
greenhouse. A beaker, magenta box, or clear top should be placed over the top of the
seedling for a week to protect it from low humidity and/or uprooting caused by
watering.
Stock solutions
A. B5 Major (10X Stock)
KNO3 (Potassium nitrate)
CaCl 2H2O (Calcium chloride)
MgSO4 7H2O (Magnesium sulfate)
(NH4)2 SO4 (Ammonium sulfate)
NaH2PO4 H2O (Sodium phosphate)
F.W.
Conc.
g/l
101.1
147.02
246.48
132.14
137.99
0.25 M
0.01 M
0.01 M
0.01 M
0.01 M
25 g/l
1.5 g/l
2.5 g/l
1.34 g/l
1.5 g/l
F.W.
Conc.
g/l
61.83
169.01
287.54
166.01
241.9
249.68
237.95
5 mM
10 mM
0.7 mM
0.45 mM
0.1 mM
0.01 mM
0.01 mM
0.3 g/l
0.76 g/l
0.2 g/l
75 mg/l
25 mg/l
2.5 mg/l
2.5 mg/l
F.W.
Conc.
g/l
180.2
123.1
205.6
337.3
0.055 M
0.8 mM
0.5 mM
3 mM
10 g/l
0.1 g/l
0.1 g/l
1 g/l
B. B5 Minor (100X Stock)
H3BO3 (Boric acid)
MnSO4 H2O (Manganese sulfate)
ZnSO4 7H20 (Zinc sulfate)
KI (Potassium iodide)
Na2MoO4 2H2O (Molybdic acid)
CuSO4 5H2O (Cupric sulfate)
CoCl2 6H2O (Cobalt chloride)
C. B5 Vitamins (100X Stock)
Myo-inositol
Nicotinic acid
Pyridoxine-HCl
Thiamine-HCl
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D. MS Major (10X Stock)
NH4NO3 (Ammonium nitrate)
KNO3 (Potassium nitrate)
CaCl2 2H2O (Calcium chloride)
MgSO4 7H2O (Magnesium sulfate)
KH2PO4 (Potassium phosphate)
F.W.
Conc.
g/l
80.04
101.11
142.02
246.48
136.09
0.2 M
0.2 M
0.03 M
0.015 M
0.0125 M
16.5 g/l
19 g/l
4.4 g/l
3.7 g/l
1.7 g/l
F.W.
Conc.
g/l
61.83
169.01
287.54
166.01
241.9
249.68
237.95
10 mM
13 mM
3 mM
0.5 mM
0.1 mM
0.01 mM
0.01 mM
0.62 g/l
2.23 g/l
0.86 g/l
83 mg/l
25 mg/l
2.5 mg/l
2.5 mg/l
F.W.
Conc.
g/l
278.0
372.24
10 mM
10 mM
2.78 g/l
3.72 g/l
E. MS Minor (100X Stock)
H3BO3 (Boric acid)
MnSO4 H2O (Manganese sulfate)
ZnSO4 7H20 (Zinc sulfate)
KI (Potassium iodide)
Na2MoO4 2H2O (Molybdic acid)
CuSO4 5H2O (Cupric sulfate)
CoCl2 6H2O (Cobalt chloride)
F. MSIII Iron (100X Stock)
FeSO4 7H2O (Ferrous sulfate)
C10H14O8Na2N2 2H2O (NaEDTA)
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