Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

1. Growth media preparation

1. For exact growth medium recipes for this protocol, please refer to Table 1.
2. For preparing 1 L of media, place a 2 L beaker on a stir plate and place a stir bar inside.
3. Fill beaker with 900 mL of distilled water and turn on the stir plate. The stir bar should be spinning at a medium speed.
4. Weigh all powdered ingredients and dissolve in a beaker.
5. Measure out all liquid ingredients, if any, and add to the beaker.
6. Bring the final volume to 1 L using distilled water.
7. Measure the pH and adjust to recipe specifications.
8. If formulating a liquid growth medium, no agar is added. Attach a filter sterilizer to a vacuum pump and pour the liquid growth medium through the filter. Turn on the pump and wait until all liquid is pulled through. Place a cap on the container and attach a label.
9. If formulating a solid growth medium, after the pH is adjusted, add agar directly into a bottle or flask.
10. Pour the 1 L liquid growth medium into a 2 L Erlenmeyer flask, or divide it into two 1 L autoclavable bottles (500 mL each). If two bottles are used, divide the agar and add directly to the bottles.
11. Cover flask with a breathable cover, such as two layers of aluminum foil, to allow steam to escape. If using a bottle, loosely cap the screw lid on the top.
12. Autoclave at 121 °C for 25 min.
13. After autoclaving, remove the growth medium from the autoclave and cool to 55-60 °C (a water bath set to 55 °C can make this easier). Keep the growth medium in a liquid state for a few hours until it is convenient to pour plates.
14. Once cooled, add all post sterilization additives (see Table 1) and mix thoroughly.
15. After all ingredients are added, pour the designated volume into the container of choice in a laminar flow hood.
16. Growth medium can be poured into desired Petri dishes manually or using a liquid dispensing apparatus. When pouring manually, it is recommended to transfer a large volume of autoclaved medium into a smaller sterile beaker (500 mL) for ease of handling.
17. Allow growth medium to cool and solidify.
18. The growth medium will be available for use once becoming solid and is best used the following day after drying slightly overnight in a sterile flow hood as stacks of lidded plates. After overnight drying, transfer the plates into plastic sleeves, fold over the loose end, and keep this in place with a small bit of tape. This prevents excessive drying. Medium can be stored in a cool, dark, and clean environment (4-16 °C) for up to 1 month.

2. Growing donor plants and harvesting immature ears

1. Grow any publicly available maize inbred (i.e., B73, Mo17, or W22) in a greenhouse in 1.5 gallon (5.9 L) pots containing a soilless substrate. Use a 16/8 (day/night) photo period, with average temperatures of 25.5 °C during the day and 20 °C at night.
2. Plants are watered as needed and fertilized with a controlled release fertilizer (N-P-K of 15-9-12), which can either be incorporated into the soil mix or added to the surface after planting.
3. It usually takes about 70-90 days after seed germination for ears to emerge. As ear shoots emerge, cover them with a shoot bag to prevent uncontrolled pollination from occurring.
4. About 2-3 days after silks have emerged and if pollen will be available the following day, cut the silks using scissors that have been sterilized in 70% ethanol. Cut the silks and husk roughly 2.5 cm below the end of the husk leaves, where the silks emerge. Pollination can be performed the next day. Be sure to resterilize scissors between each ear.
5. Once anthers emerge from a tassel, cover the tassel with a tassel bag and non-skid paper clip at the base of the bag around the stalk.
6. The next morning, gently bend the plant over and tap the bag to encourage pollen to be released.
7. Remove the tassel bag and fold the top of the bag over to prevent pollen from escaping. It is generally best to bag the tassel 1 day before it will be used (to avoid build-up of dead pollen and shed anthers). Fresh pollen may be collected from tassels for about 3-5 days. When anthers emerge from the inner florets at the base of the tassel, that tassel will likely not produce viable pollen the next day.
8. Use the pollen from the same plant (selfing) or from another plant of the same inbred (sibbing).
9. Remove the ear bag or cut the end of the bag to expose the silks, then quickly pour pollen from the tassel bag onto the silks.
10. Cover the ear with the tassel bag immediately and staple the base of the bag around the stalk to secure it. It may be helpful to physically isolate the plant from flowering plants of different genotypes during pollination to help prevent cross-pollination. Leave the tassel bag on the ear until the immature ear is ready to harvest.
11. 9-12 days after pollination, screen ears for embryo size. Slide the pollination bag up the stalk to expose the ear. Gently pull the husk down to expose kernels on about one-third to one-fourth of the circumference of the ear and about one-third of the distance down the ear. Kernels near the tip will not be representative of the average embryo size.
12. Using a scalpel, slice off the cap of a single kernel that appears similar to the majority of other kernels in size and color.
13. Use a spatula (with a ruler) to remove the embryo as described in step 4.6. Measure the length of the embryo using a built-in ruler on the spatula or a digital caliper. If the embryo is between 1.5-2.0 mm, harvest the ear. If it is ~1.3 mm, the ear may be ready to harvest later in the day and can be checked again in about 7-8 h.

3. Preparing Agrobacterium suspension culture for infection

NOTE: The Agrobacterium strain LBA4404(Thy-) containing PHP81430 (Figure 1) and PHP71539¹² is stored as a glycerol stock at -80 °C. These materials can be obtained from Corteva Agriscience through a Material Transfer Agreement. LBA4404(Thy-) is an auxotrophic strain that needs thymidine supplied in the growth media. The primary utility of the auxotroph Agro strain is for biocontainment purposes. It has the additional benefit of reducing Agro overgrowth. The auxotrophic Agro strain does not grow without supplemental thymidine. Nevertheless, thymidine can (presumably) be supplied by dying plant tissue in the culture. Therefore, there is still a need to provide an antibiotic in the medium to completely control the auxotrophic Agro. However, it will be easier to control due to compromised growth of the auxotrophic strain in the absence of thymidine.

1. Four days before the date of infection, initiate a "mother" plate from the glycerol stock by streaking the bacteria on a YP plate with 50 mg/L thymidine, 50 mg/L gentamicin, and 50 mg/L spectinomycin (Table 1). Incubate the "mother" plate in a 20 °C incubator for 3 days.
2. One day before the infection experiment, prepare a "working" plate by selecting one to five colonies from the "mother" plate and streaking the bacteria from the "mother" plate to a new YP plate (with thymidine, gentamycin, and spectinomycin; Table 1).
3. Streak the daily "working" plate in sequential quadrants and run the loop 1x through the just-streaked area into the successive quadrant, repeating to form quadrants that have been serially diluted. Incubate the "working" plate overnight in a 27 °C incubator.
4. After completing embryo dissection (step 4.8), use a loop or similar tool to collect Agrobacterium from a region of the "working" plate where bacterial growth is visible as thin streaks of colonies.
   NOTE: Avoid areas of the plate with a dense lawn of bacterial growth. The Agrobacterium growth has likely already started to decline in dense areas, while in the areas with visible colonies the bacteria are in the proper growth phase for infection.
5. Suspend the collected bacteria in a 50 mL tube containing 10 mL of 700A liquid medium (Table 1). Vortex to suspend the bacteria culture completely.
6. Measure the optical density at a wavelength of 550 nm. Adjust the volume until the OD is between 0.35-0.45, with 0.4 being the optimal value.
   NOTE: If the OD is higher than 0.45, add more 700A liquid medium. If the OD is lower than 0.35, inoculate more Agro colonies in the suspension culture.

4. Embryo dissection, infection, and co-cultivation

1. Select suitable ears for transformation experiments; these should have a good seed set and have embryos that range in size from 1.5-2.0 mm. They are typically harvested between 9-12 days after pollination. Harvested ears can be used fresh or stored for 1-4 days at 4 °C, though quality of response will likely degrade progressively with prolonged storage beyond the first day.
2. Remove the husks and silks. Insert a handle into either the base or the top of the ear. The handle can be a pair of forceps, screwdriver, etc.
3. Place ears in a large container (e.g., 2 L beaker with the handle upwards, fill the container with disinfection solution. Disinfection solution is 1.8 L of 20% commercial bleach (1.65% sodium hypochlorite) and a couple of drops of surfactant Tween 20.
4. Sterilize the ears inside a laminar flow bench. After 20 min, empty the bleach solution and rinse the ears 3x (5 min each) using a generous amount of sterile distilled water. Remove the water and allow ears to dry for several minutes.
   NOTE: It is important that the ears be completely submerged in the bleach solution for 20 min. Move the ears carefully around in the bleach solution occasionally to dislodge air bubbles.
5. Prepare a 2 mL microcentrifuge tube filled with 700A liquid medium. This tube will be used to collect the immature embryos.
6. Take the ear and using a sterile scalpel, remove the top 1-2 mm of the kernel crowns to expose the endosperm. Use a micro spatula to remove the immature zygotic embryo (IZE). The IZE will be located within the kernel, on the side facing the tip of the ear, and near the attachment to the cob. Using the spatula, insert it into the endosperm in the pericarp furthest away from the embryo, then gently twist upward to dislodge the endosperm and allow for removal of the embryo (Figure 2).
7. Using the spatula, transfer the embryo into the tube containing the 700A liquid medium. Continue doing this until up to 100 embryos have been collected. Multiple tubes may be filled (~100 embryos/tube) before proceeding to the next step. At this point, the Agrobacterium suspension should be prepared (see step 3.5).
8. Remove the 700A liquid medium from the embryo tube with a 1 mL pipette. Add fresh 700A medium to wash the embryos, then remove that media as well.
9. Add 1 mL of the Agrobacterium suspension and vortex on a low setting (3/10) for 30 s or invert tube 12x-15x to mix. Allow this tube to rest horizontally on bench for 5 min.
10. After 5 min, transfer the entire tube of embryos and Agrobacterium suspension onto a plate of 562V co-cultivation medium (Table 1). This can be achieved by placing the plate on the bench and quickly pouring the tube contents onto the plate. Gently swirl the plate to distribute the embryos and remove the Agrobacterium suspension using a 1 mL pipette.
11. Make sure that the embryos are placed with the scutellum (round) side facing upwards. Use a magnifying glass or a dissecting scope, if needed. Place plates in plastic boxes (19 cm x 28 cm x 5.1 cm) and incubate the plates overnight 16-18 h at 21 °C in the dark. No individual plate wrapping using paraffin film or vent tape is necessary.
12. After overnight co-cultivation, move the infected embryos, scutellum side up, onto resting medium 605T (Table 1). Place around 30 embryos per plate. Incubate the plates at 26-28 °C in the dark.
13. Incubate for 4-10 days (7 days is preferred). At this time, the development of somatic embryos can be observed on the surface of the zygotic scutellum (Figure 3).

5. Selection, heat treatment, and regeneration

1. After the resting period, heat shock the embryos. Place the box containing the plates of embryos in a 45 °C incubator with 70% relative humidity for 2 h. Then, remove the box from the 45 °C incubator and place in the 26-28 °C dark incubator for 1-2 h.
   NOTE: If unable to attain 70% humidity in an incubator, add a double layer of autoclaved paper towels to the bottom of the plate box and soak with autoclaved water to maintain humidity within the box. Return the plates to the box on top of the paper towels and seal the lid before placing at 45 °C. Use a small digital hygrometer/thermometer to monitor the temperature and humidity.
2. Transfer the heat-treated IZEs from the resting medium to the shoot formation medium (13329A) containing 0.05 mg/L imazapyr as a selective agent (Table 1). When transferring, remove coleoptiles, if present, using fine tip forceps or surgical scissors.
3. Place 10-15 embryos per plate to avoid overcrowding. Keep the embryos in this medium for 2 weeks in the 26 °C dark incubator.
4. Transfer the embryos to rooting medium (13158; Table 1) for 1-2 weeks. Place around eight pieces per plate and incubate in a light room or light chamber (16 day/8 night, 20-150 µmol/m²/s) at 27 °C.
5. As plantlets develop, place stronger plantlets containing both shoots and vigorous roots onto new plates of rooting medium, place one plant per plate. This will allow for stronger plantlet growth. Place the plates in the light room or light chamber for another 7-14 days.
   NOTE: Carefully remove any callus pieces associated with the plantlet to ensure it is in good contact with the medium.
6. As the plant becomes more vigorous, remove the plant from rooting medium and rinse the roots with tap water to remove agar.
7. Transplant individual plants into 3 in² (~19 cm²) pots containing a pre-wetted soilless substrate. Place the pots in a tray (27 cm x 54 cm) with drain holes and cover the flat with a plastic humidity dome. This acclimation step can be achieved either in growth chamber or in greenhouse with growth conditions described in section 2 (step 2.1) above.

6. Transplanting to the greenhouse and the production of T1 seeds

1. Check the plants 2x per day. Water as needed. Ensure that the plants are neither dried out nor overwatered. Maintaining a slightly dry substrate encourages root growth.
   NOTE: The humidity dome can be removed 4-7 days after transplanting. Plants should be grown in these small pots until they have visibly recovered from the stress of transplant to soil. This should take about 9-14 days.
2. Transplant the entire soilless plug and plantlet into a 1.5 gal (5.9 L) pot. Maintain in the greenhouse and water when soil feels dry to the touch.
3. Add a controlled release fertilizer with N-P-K of 15-9-12 to the pot, which can be either incorporated into the substrate mix or applied to the surface.
4. When ear shoots begin to emerge from the plant, use a shoot bag to cover the ear shoots. Be sure to use a bag that is semi-transparent so that the emerging silks can be observed without removal of the bag. The shoot bag allows for controlled pollination to occur. It is important to always bag transgenic tassels.
5. After the silks emerge (1-2 days), trim the emerged silks to a uniform length. This will be about 2.5 cm below the top of the husk leaves. Use a pair of clean scissors that have been sterilized in 70% ethanol. By trimming the silks, a uniform tuft develops the following day for pollination to occur.
6. For a majority of maize genotypes, the optimal time for pollination is 2-3 days after tassel or silk emergence.
7. Collect the pollen from either the same plant (if being self-pollinated) or from a wild-type of the same inbred (if outcrossing or incrossing).
8. Collect the pollen in a tassel bag and apply it to the tuft of silk of the T0 plant. If the pollen is from a wild-type (non-transgenic) plant, place the pollen in a plain brown tassel bag. If the pollen is from a transgenic plant, place the pollen in a green striped bag to indicate that the pollen is transgenic.
9. Follow steps 2.5-2.10 for pollination details.
10. About 2 weeks after pollination, remove the tassel bags from ears, and allow for dry-down to begin. To help dry down, stop watering the plant 21-25 days after pollination. You can also pull back the husk leaves to expose the seed. This practice also helps prevent mold.
11. About 45 days after pollination, harvest seeds and store in cold storage at 4-12 °C.