Soybean Cotyledonary-node Agrobacterium-mediated Transformation System
The protocol outlined below is a modification of that described by Hinchee et al. (Bio/Technology 1988. 6:915). The modifications have been previously reported. See Zhang et al., 1999.
Plant Cell Tiss. Organ Cult. 56:37; Clemente et al. 2000. Crop Sci. 40:797, and Xing et al. 2000 In Vitro Cell Dev. Biol. Plant 36:456.
· Seed Sterilization Step
We currently use three genotypes A3237 (Asgrow Seed Company), Thorne (Ohio State University) and NE3001 (University of Nebraska).
Place seed in an open petri plate as a single layer. Place the petri plate into a standard size desiccator within a fume hood. Place a 250 ml beaker with 100 ml of Chlorox™ bleach in the center of the desiccator. Add 3.3 ml of 12 N HCl, dropwise, along the side of the beaker. Close the desiccator and let stand overnight. The next day cover petri plate with a sterile lid. The sterilization may need to be repeated depending on the seed lot.
· Seed Germination Step
Germinate the sterilized seeds on germination medium (GM) for 5 days at 24°C (18/6) light regime) in 100 X 25 mm petri plates (Place 10-15 seed per plate). Stack the plates 5 high and place in a clear plastic bags with 4 to 5 slices in the bag. Seed Germination.
GM medium
Gamborg's B5 medium supplemented with 2% sucrose, pH 5.8, solidified with 0.8% agar.
· Agrobacterium Inoculum Preparation>
The protocol outlined below is a modification of that described by Hinchee et al. (Bio/Technology 1988. 6:915). The modifications have been previously reported. See Zhang et al., 1999. Plant Cell Tiss. Organ Cult. 56:37; Clemente et al. 2000. Crop Sci. 40:797, and Xing et al. 2000 In Vitro Cell Dev. Biol. Plant 36:456.
Pellet 35 ml of the A. tumefaciens culture at 3,000 to 5,000 rpm for 10 min. Suspend the bacterial pellet in co-cultivation medium to a final OD650 of 0.6 to 1.0.
Co-cultivation Medium
1/10th Gamborg's B5 medium supplemented with 1.67 mg/l BAP, 0.25 mg/l GA3, 3% sucrose, 200 μM acetosyringone, 20 mM MES, pH 5.4. Filter sterilize all growth regulators and acetosyringone.
· Explant Preparation
Carefully check the germination plates for any signs of contamination. Discard all contaminated plates. Only select seedlings> that are green and healthy in appearance. Excise the germinating seed from the root system by making a cut through the hypocotyl region. With a scalpel blade cut the seed vertically through the hypocotyl region. Remove the embryonic axis tissue that remains attached to the cotyledon.
Make 7 to 12 vertical slices, parallel to the axis, about the junction between the cotyledon and the hypocotyl. Slices should be approximately 3 to 4 mm long, with sufficient wounding to prevent the axial shoot from emerging, yet not impede de novo shoot production. Prepare 30 to 40 explants (an explant is one cotyledon with hypocotyl attached, i.e. two explants per seed) and initiate inoculations. While the first set of explants is inoculating begin to prepare more explants
· Explant Inoculation
Place 25 ml of Agrobacterium inoculum in to a petri plate. Add the 30 to 40 prepared explants, be sure explants are completely covered with the inoculum. The inoculation step should be carried out for 30 min., with occasional agitation. Place the explants on co-cultivation plates (5 per plate), adaxial side down.
· Co-cultivation Plates
Co-cultivation medium solidified with 0.5% purified agar overlay a piece of sterile filter paper on the top of each plate. Wrap the plates with parafilm and place at 24°C, 18/6 ligh regime. Allow the tissue to co-cultivate for 3 days. Following the co- cultivation period briefly was the explants in liquid shoot initiation medium supplemented with 0.25 mg/l GA3
Pellet 35 ml of the A. tumefaciens culture at 3,000 to 5,000 rpm for 10 min. Suspend the bacterial pellet in co-cultivation medium to a final OD650 of 0.6 to 1.0.
Shoot Initiation Medium
Gamborg's B5 medium supplemented with 1.67 mg/l BAP, 3% sucrose, 50 mg/l ticarcillin, 50 mg/l cefotaxime, 50 mg/l vancomycin; 3 mM MES, pH 5.6. Solidify medium with 0.8% purified agar. If using the bar gene as a marker use 5 mg/l glufosinate. Filter sterilize all growth regulators, vitamins, antibiotics and selection agent.
· Shoot Initiation Step
Culture the explants adaxial side up, with the junction of the hypocoyl/cotyledon jut below the surface. Wrap the plates (100 X 25 mm) with porous tape and culture at 24°C, 18/6 light regime.
Transfer the tissue to fresh shoot initiation medium after 14 days. At the 124-day period cut the hypocoyl region flush to the developing node. Orient the tissue so that the freshly cut surface is imbedded in the medium, with the differentiating region flush to the surface. Allow the tissue to culture for an additional two weeks under the same environmental conditions.
· Shoot Elongation Step
At the end of the shoot initiation period only carry on the differentiating explants and discard the rest. Approximately 80% of the explant should be differentiating at this point.
Remove the cotyledon from the explants, make a fresh cut at the base of the developing node (horizontally) and transfer the tissue to shoot elongation medium. Culture at 24°C (18/6 light regime)
Shoot Elongation Medium
MS salts/Gamborg's vitamins supplemented with 1 mg/l zeatin riboside, 0.1 mg// IAA, 0.5 mg/l GA3, 3% sucrose, 100 mg/l pyroglutamic acid, 50 mg/l asparagine, 3 mM MES (pH 5.6) solidified with 0.8% purified agar. Maintain the levels of ticarcillin, cefotaime and vancomycin to that used during shoot initiation. If using the bar gene as a marker use 3 mg/l glufosinate. Filter sterilize all growth regulators, vitamins, antibiotics and selection agent.
Transfer the tissue to fresh shoot elongation medium every two weeks. At each transfer make a fresh horizontal slice at the base of the tissue. Once a shoot has elongated to approximately 1 cm in size transfer the isolated elongated shoot to a cup with elongation medium for an additional two weeks.
· Rooting Step
Root elongated shoots (> 3 cm) on rooting medium without further selection.
Rooting medium
1/2 MS salts full strength Gamborg's vitamins, 2% sucrose, 0.5 NAA, 50 mg/l asparagine, 100 mg/l pyroglutamic acid, 3 mM MES (pH 5.6). Solidify the medium with 0.8% purified agar. Do not supplement the medium with antibiotics used to counter select A. tumefaciens. This will drastically impede root development. Root the shoots in cups. Rooting usually takes between 2 to 4 weeks. Acclimate the rooted shoots in Metro Mix 360 prior to establishing in the greenhouse.
