Improvement of Agrobacterium and Soybean Use

Soybeans (Glycine max) are a globally important source of livestock food, biofuels, oils, and direct food products. Despite soybeans comprising 50% of all bioengineered crops¹, there have been no iGEM teams who have bioengineered Glycine max. We think beans are the best, and hope to see future iGEM teams fully utilize this mighty plant to its full potential. As our contribution to iGEM and its future teams, we have established a protocol for agrobacterium-mediated plant transformation.

Agrobacterium (A. tumefaciens) is much tricker to work with than E. coli, and traditional agrobacterium-mediated tissue cultures of plants involve weeks of subculturing on many different media. This attached protocol is an improvement upon many others, especially for groups with time sensitive needs such as iGEM teams. It is particularly engineered to work with EHA 105s and soybean cultivars Williams 82 and Maverick.

A simplified version of this protocol is also available on our Experiments page.

Plant Transformation Protocol

Traditionally agrobacterium-mediated transformation was a long and technical process involving repeated subculturing of plant tissue across four different media over the course of 16 weeks before transfer to a greenhouse for traditional cultivation. We have eliminated 75% of this subculture by optimizing the screening method and relying on the cotyledon as an energy source for the transgenic shoots (the energy sink). This protocol was originally engineered by our lab manager Simon Kalmus and practiced with the modifications of YEP for AB Minimal and addition of reducing agents mentioned below for the composite plasmid CrtE-cytoTDS-MBP_RUBY. It should be noted that it has been designed for EHA 105s and soybean cultivars Williams 82 and Maverick. Different cultivars and species will respond best to different strains of agrobacterium.

Day 1, Morning

Buffers are made. YEP medium may be substituted for AB Minimal Medium.

1. Autoclave many paper towels, 1 70mm Whatman filter paper per 10 beans, 1 baffled 250mL culture flask, and roughly 30mL DDH2O.
2. Prepare filter sterilized AB Minimal Medium with 10.21 g/L AB minimal medium powder in a 250mL beaker, stir until dissolved. With a Pall filter, filter sterilize into an autoclaved and cooled baffled flask.
3. Make and autoclave your Infection medium.
   • Gamborg B5 Basal Medium (Refrigerated): 3.21g/L
   • Sucrose: 30g/L
   • MES: 3.9g/L
   • Titrate to pH 5.4 with 1 M KOH
4. Make and autoclave the Co-cultivation medium (100mL per 40-60 explants or 20-30 beans)
   • Gamborg B5 Basal Medium (Refrigerated): 3.21g/L
   • Sucrose: 30g/L
   • MES: 3.9g/L
   • 7g/L Noble Agar
   • Titrate to pH 5.4 with 1M KOH
5. Leave the Infection medium and co-cultivation medium to cool at room temperature overnight in preparation for day 3

Day 1, ~5pm

The sterilized beans are imbibed, and the agrobacterium liquid culture is created. YEP medium may be substituted for all directions indicating use of AB Minimal medium; the only difference is your agrobacterium will grow faster in YEP when compared to AB minimal.

1. Under the laminar flow hood: completely submerge the sterilized beans with sterile dH2O in the 15x100mm plate.
2. Cover with foil and leave on the bench.
3. Set aside 1mL of the filter sterilized AB minimal medium (OR YEP Medium) in a clean cuvette to blank the spectrophotometer with on day three.
4. Inoculate the AB minimal medium with 100-300uL of the saturated 5mL YEP culture.
5. Shake this medium overnight at ~220rpm at 28 ℃.

Day 2

The actual tissue culture is started. Everything needs to be done as aseptically as possible and needs to occur under a laminar flow hood.

OD 600 Reading

1. In the morning, allow the spectrophotometer to warm up and blank it with the sample of AB minimal allocated yesterday.
2. Check the OD600 of the AB minimal culture, hopefully it is at OD600 0.6A.

Spindown and Resuspension in Infection Medium

1. If the OD600 of the AB minimal culture is already above 0.6A, perform a 2x dilution with ddH2O and reread. Using this diluted reading, perform the math needed to resuspend your culture at an OD600 of 0.6A. Spin down the necessary amount so that a resuspended volume of 35mL of cells would have an OD of 0.6
2. In the laminar flow hood, add the following to your infection medium at the correct concentrations
   • 200uM Acetosyringone (100mg/mL filter-sterilized aliquots)
   • 1.67mg/L Benzyl-aminopurine (30mg/mL filter sterilized aliquots)
   • 0.25mg/L Gibberellic Acid (32mg/mL filter sterilized aliquots)
   • Optionally, include 40mg cystine and 100uL 100X 1,4-Dithiothreitol per 100mL medium
3. In a falcon tube, resuspend the spun down pellet in roughly 10mL of infection medium. VERY GENTLY pipette up and down to resuspend the pellet. These cells need to remain alive.
4. Add the remaining infection medium needed to bring the volume of the suspension to 35mL.
5. Place falcon tube in the 28 ℃ incubator for 30 minutes shaking gently.

Soybean Explant Preparation and Infection
We have been using a similar method described in Pareddy et. Al (2020) for our explant preparation. This leaves most of the embryonic tissue present and intact for infection by the agrobacterium.

1. Wipe down your scalpel with 70% ethanol in the laminar flow hood.
2. Cut the bean longitudinally starting in the middle of the hypocotyl.
3. Cut to about halfway down the bean.
4. Gently pinch the bean to separate the seed coat.
5. Cut most of the radical off.
   You should now have two explants from each plant that look like this:
   
6. Separate the two explants and add them directly to the infection medium.
7. Once all explants are prepared and added to the infection medium, seal the falcon tube and place it on its side in the laminar flow hood.
8. Gently roll the falcon tube back and forth to swirl the beans around in the tube. Let it sit for 30 minutes, occasionally rolling to agitate.

Co-cultivation Plate Preparation
While your beans are in the infection medium, you can prepare your plates.

1. Remelt the co-cultivation medium in the microwave. DO NOT have the top of this bottle off outside of the laminar flow hood.
2. Once melted, inside of the laminar flow hood, add the following to the cocultivation medium at the correct concentrations.
   • 200uM Acetosyringone (100mg/mL filter-sterilized aliquots)
   • 1.67mg/L Benzyl-aminopurine (30mg/mL filter sterilized aliquots)
   • 0.25mg/L Gibberellic Acid (32mg/mL filter sterilized aliquots)
   • Optionally, include 40mg cystine and 100uL 100X 1,4-Dithiothreitol per 100mL medium
3. Pour your plates under the hood and leave to cool.
4. Once solidified, place one whatman filter paper on top of the medium in each plate and allow to settle.
5. Leave plates inside of the laminar flow hood for the plating of transformed beans.

Plating the Transformed Beans

1. Wipe down tweezers with 70% ethanol, allow to dry.
2. Using the tweezers, pull explants out of the falcon tube and place flat side down on the autoclaved paper towels to wick away axess agrobacterium.
3. Once all of the explants have been pulled out, one at a time and with the teezers, flip an explant on the paper towel to dry the other side and place flat side down on the filter paper plates.
   • If the paper towel becomes too saturated with theinfection medium, replace with a fresh one.
4. Plate 8-10 explants per plate.
5. Seal the plates with sterilized parafilm and place in the incubator for 5 days of cocultivation.

The plated beans should look similar to this immediately after plating.



Within a day or two, the beans should begin to turn green and swell. The embryonic tissue will begin to grow.

Day 7

After 5 days, the beans are ready to be rinsed and subcultures on a Shoot Induction Medium (SIM)

By day 7 of the plant transformation protocol, the tissue explants should be fully green and show signs of growth from embryonic tissues (hypocotyl and epicotyl).

1. Prepare your mediums
• 500mL ddH2O in a beaker, cover with foil
• 2 x 250mL rinsing medium in separate beakers, cover with foil
• Gamborg B5 Basal Medium (Refrigerated): 3.21g/L
• MES: 600mg/L
• Titrate to pH 5.7 with 1M KOH
• Solid SIM (~30mL/tissue culture vessel, 6 explants per vessel, or about 500mL/100 explants)
• Gamborg B5 Basal Medium (Refrigerated): 3.21g/L
• MES: 600mg/L
• 7g/L Noble Agar
• Titrate to pH 5.7 with 1M KOH
• Prepare the following antibiotics and then filter sterilize these under the fume hood
• Cefotaxime stock 200mg/6mL ddH2O
• Vancomycin stock 50mg/2mL ddH2O
• Timentin stock 100mg/2mL ddH2O
2. Autoclave
• After autoclaving these mediums, everything needs to be done in sterile conditions in the laminar flow hood
3. To the SIM and rinsing media, add the following antibiotics and benzo(a)pyrene (BAP) at the concentrations mentioned
• Cefotaxime 200mg/6L ddH2O
• Vancomycin 50mg/2L ddH2O
• Timentin 100mg/2L ddH2O
• 1.11 mg/L BAP (30mg/mL filter sterilized stock)
4. Pour your tissue culture vessels at 16 culture vessels/L SIM

Rinsing and Subculturing

1. With tweezers in the laminar flow hood, take each explant and serially wash them through the ddH2O, rinsing medium beaker 1, rinsing medium beaker 2. Place on sterile paper towel to dry.
2. Proceed with rinsing the explants until you have rinsed all of the explants from one plate. Place curved side of explant down, with the embryonic node flush with the SIM medium, 6 explants per tissue culture vessel