We thank the Corteva greenhouse team for providing maize immature ears, the Corteva media prep lab for providing assistance in making media, Ning Wang from Corteva for help with the Agrobacterium construct, and Keunsub Lee from Iowa State University for assistance. This project was partially supported by National Science Foundation Plant Genome Research Program Grant 1725122 and 1917138 to K.W., by Predictive Plant Phenomics Research Traineeship Program (National Science Foundation Grant DGE-1545453) to J.Z., by the USDA NIFA Hatch project #IOW04341, by State of Iowa funds, and by Crop Bioengineering Center of Iowa State University.

1. Growth media preparation
<ol>
	<li>For exact growth medium recipes for this protocol, please refer to Table 1.</li>
	<li>For preparing 1 L of media, place a 2 L beaker on a stir plate and place a stir bar inside.</li>
	<li>Fill beaker with 900 mL of distilled water and turn on the stir plate. The stir bar should be spinning at a medium speed.</li>
	<li>Weigh all powdered ingredients and dissolve in a beaker.</li>
	<li>Measure out all liquid ingredients, if any, and add to the beaker.</li>
	<li>Bring the final volume to 1 L using distilled water.</li>
	<li>Measure the pH and adjust to recipe specifications.</li>
	<li>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.</li>
	<li>If formulating a solid growth medium, after the pH is adjusted, add agar directly into a bottle or flask.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Autoclave at 121 &#176;C for 25 min.</li>
	<li>After autoclaving, remove the growth medium from the autoclave and cool to 55-60 &#176;C (a water bath set to 55 &#176;C can make this easier). Keep the growth medium in a liquid state for a few hours until it is convenient to pour plates.</li>
	<li>Once cooled, add all post sterilization additives (see Table 1) and mix thoroughly.</li>
	<li>After all ingredients are added, pour the designated volume into the container of choice in a laminar flow hood.</li>
	<li>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.</li>
	<li>Allow growth medium to cool and solidify.</li>
	<li>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 &#176;C) for up to 1 month.</li>
</ol>
2. Growing donor plants and harvesting immature ears
<ol>
	<li>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 &#176;C during the day and 20 &#176;C at night.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>The next morning, gently bend the plant over and tap the bag to encourage pollen to be released.</li>
	<li>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.</li>
	<li>Use the pollen from the same plant (selfing) or from another plant of the same inbred (sibbing).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Using a scalpel, slice off the cap of a single kernel that appears similar to the majority of other kernels in size and color.</li>
	<li>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.</li>
</ol>
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 &#176;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.
<ol>
	<li>Four days before the date of infection, initiate a &#34;mother&#34; 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 &#34;mother&#34; plate in a 20 &#176;C incubator for 3 days.</li>
	<li>One day before the infection experiment, prepare a &#34;working&#34; plate by selecting one to five colonies from the &#34;mother&#34; plate and streaking the bacteria from the &#34;mother&#34; plate to a new YP plate (with thymidine, gentamycin, and spectinomycin; Table 1).</li>
	<li>Streak the daily &#34;working&#34; 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 &#34;working&#34; plate overnight in a 27 &#176;C incubator.</li>
	<li>After completing embryo dissection (step 4.8), use a loop or similar tool to collect Agrobacterium from a region of the &#34;working&#34; 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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Embryo dissection, infection, and co-cultivation
<ol>
	<li>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 &#176;C, though quality of response will likely degrade progressively with prolonged storage beyond the first day.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare a 2 mL microcentrifuge tube filled with 700A liquid medium. This tube will be used to collect the immature embryos.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C in the dark. No individual plate wrapping using paraffin film or vent tape is necessary.</li>
	<li>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 &#176;C in the dark.</li>
	<li>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).</li>
</ol>
5. Selection, heat treatment, and regeneration
<ol>
	<li>After the resting period, heat shock the embryos. Place the box containing the plates of embryos in a 45 &#176;C incubator with 70% relative humidity for 2 h. Then, remove the box from the 45 &#176;C incubator and place in the 26-28 &#176;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 &#176;C. Use a small digital hygrometer/thermometer to monitor the temperature and humidity.</li>
	<li>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.</li>
	<li>Place 10-15 embryos per plate to avoid overcrowding. Keep the embryos in this medium for 2 weeks in the 26 &#176;C dark incubator.</li>
	<li>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 &#181;mol/m2/s) at 27 &#176;C.</li>
	<li>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.</li>
	<li>As the plant becomes more vigorous, remove the plant from rooting medium and rinse the roots with tap water to remove agar.</li>
	<li>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.</li>
</ol>
6. Transplanting to the greenhouse and the production of T1 seeds
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>For a majority of maize genotypes, the optimal time for pollination is 2-3 days after tassel or silk emergence.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Follow steps 2.5-2.10 for pollination details.</li>
	<li>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.</li>
	<li>About 45 days after pollination, harvest seeds and store in cold storage at 4-12 &#176;C.</li>
</ol>

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+Tn21,+flagship+of+the+floating+genome.">Transposon Tn21, flagship of the floating genome.</a> Microbiology and Molecular Biology Reviews. 63 (3), 507-522 (1999).</li><li>Nishiguchi, R., Takanami, M., Oka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+sequence+determination+of+the+replicator+region+in+the+hairy-root-inducing+plasmid+pRiA+4b.">Characterization and sequence determination of the replicator region in the hairy-root-inducing plasmid pRiA 4b.</a> Molecular and General Genetics. 206 (1), 1-8 (1987).</li></ol>

Alicia Masters, William Gordon-Kamm, and Todd Jones are employees of Corteva Agriscience that supplied the protocol and maize ears of B73, Mo17, and W22 used in this article. The authors Minjeong Kang, Morgan McCaw, Jacob Zobrist, and Kan Wang have nothing to disclose.

Traditional protocols for maize transformation follow the paradigm of isolating immature zygotic embryos to produce transgenic callus tissue, which is regenerated into fertile plants4,6. While this is effective, callus-based protocols can be time-consuming, and it often takes up to 3 months for the tissue culture process to produce plants. What makes the method presented here significant is that it is callus-free, efficient, quick, and allows for the regeneration of T0 plants in roughly half the timeframe. It also appears to be less genotype-dependent and can thus be effective for most publicly available inbreds8,11.
While all steps should be effectively followed, correct growth media preparation is imperative. Growth media components need to be added at the correct stages, both pre- and post- autoclave, to ensure that the plant material receives the proper concentration of chemicals. This will ensure that sensitive compounds like antibiotics do not break down. It is also important that plant material is placed on the correct growth medium at each stage, as indicated in the protocol. Not placing material on the proper growth medium can result in material death. In addition, placing too many embryos or developing tissues on plates should be avoided. While placing twice as many tissue pieces may save the cost of chemicals and Petri dishes (and even incubator space), the growth of tissue in overcrowded plates can be seriously inhibited. While performing the infection, it should be ensured that the optical density of the Agrobacterium suspension is appropriate. If the bacterial suspension density is too low, proper infection may not occur.
The quality of starting materials is essential for success in transformation protocols. Ears used for embryo dissection must be healthy, meaning that the plant that produces them is healthy. They also must possess an adequate seed set and be pest- and disease-free. Also, old Agrobacterium should not be used. The &#34;mother&#34; plate should be no more than 2 weeks old. After this point, a new &#34;mother&#34; plate should be streaked to begin new experiments.
While this method has been shown to be less genotype-dependent, it cannot be assumed that all lines will be equally successful. There can still be variation amongst lines as well as differences in success based upon the construct being used. Ear-to-ear variability is also unavoidable when working with immature embryos, so ideally experiments should use multiple ears to account for this. In this work, inbred W22 performed the best, with over ~14% transformation frequency, followed by B73 and Mo17 (~4% each). Lowe et al.8 reported using the QuickCorn protocol for B73 and Mo17 transformation. In this work, the transformation frequencies ranged from 9%-50% for B73 and 15%-35% for Mo17.
One possibility for the lower transformation frequencies for B73 and Mo17 observed in this work may be attributed to seasonal ear quality fluctuation. Another difference between this work and that of Lowe et al.8 is that different vector constructs were used here. In Lowe&#39;s work, morphogenic genes were not removed from the transformed plants but rather developmentally silenced in the later stages. In this work, the morphogenic genes were removed 8 days after the infection. It is possible that B73 and Mo17 may need a longer presence of Bbm/Wus2 for the development of somatic embryos.
Using this method, there is a possibility of obtaining non-transgenic escape plants, multimeric insertions, and unexcised transgenes. These plants will not have a noticeably different phenotype, so detection by PCR is required to determine whether a plant is transgenic. To accomplish this, PCR primers within the excised region and primers flanking the excised region can be employed. Multiple independent transformations can also produce plants from the same immature embryo, making determination of total independent transformant recovery rate difficult. Our standard has been to calculate a transformation rate based on sampling one plant from each immature embryo that produced plants and dividing this by the number of embryos infected. This method almost certainly underestimates the actual number of independent events recovered as plantlets. Discrimination between independent events from the same embryo requires sequencing border regions around transgenes, and this will be prohibitively expensive and time-consuming for most applications; though, there may be cases in which these data are useful.
This method of tissue culture transformation has proven to be very effective, but problems can still occur. If plant material is not responding, it is possible that there is an issue with the particular inbred line, suggesting that variables such as growth media composition and timing of subculturing require adjustments. Another variable is proper vector design and accurate vector construction, if the original vector is altered. There can also be issues with imazapyr sensitivity, as some lines are more sensitive than others, and the concentration of imazapyr may need to be adjusted to achieve successfully transformed plants.
Over the last 30 years, maize tissue culture and transformation protocols have changed and progressed; and it is believed that this shortened protocol will further this progression. This method is effective for academic settings because it is less time-consuming than traditional methods. In addition, it does not demand highly trained operators, making it more amenable to widespread distribution when compared to traditional methods. In the future, this method can be combined with new technologies such as genome engineering.

Transformation is a basic tool for evaluating foreign gene expression in maize and producing genetically modified corn lines for both research and commercial purposes. Access to high throughput transformation can facilitate the increased need for maize molecular and cellular biology studies1. The ability to genetically transform crop species is vital to both public and private laboratories. This allows for both fundamental understanding of gene regulation mechanisms as well as crop improvement on a global scale to support an ever-growing population.
The discovery that immature embryos from maize could be used for the production of regenerable callus originated in 19752. Since this revelation, most scalable maize transformation protocols have required callus formation and selection prior to regeneration3. During the process of genetic transformation, Agrobacterium-infected or biolistic-bombarded immature embryos are cultured on media for embryogenic callus induction. Induced calli are then cultured on selective media (e.g., containing an herbicide) so that only transformed callus pieces are able to survive. These herbicide-resistant putative transgenic calli are bulked up and regenerated into plants. While this method is effective, the process is long and labor-intensive, and it can take upwards of 3 months to complete4. More importantly, conventional maize transformation protocols possess a much larger limitation, that is, only a limited number of maize genotypes can be transformed5,6.
Lowe et al.7,8 previously reported a &#34;QuickCorn&#34; transformation method that not only greatly reduced the duration of the transformation process but also expanded the list of transformable genotypes. The QuickCorn method utilizes maize orthologs (Zm-Bbm and Zm-Wus2) of the Arabidopsis transcription factors BABY BOOM (BBM)9 and WUSCHEL (WUS)10. When incorporated in the transformation vector system, these genes work synergistically to stimulate embryogenic growth7.
The QuickCorn protocol described in this work was based on the protocol in Jones et al11, which was a further improvement of the method reported by Lowe et al7,8. In the present study, an Agrobacterium strain LBA4404(Thy-) harboring a binary vector construct PHP81430 (Figure 1) and accessory plasmid PHP7153912 are used for transformation. The T-DNA of PHP81430 contains the following molecular components. (1) The transformation selective marker gene Hra expression cassette. The maize Hra (Zm-Hra) gene is a modified acetolactase synthase (ALS) gene that is tolerant to ALS-inhibiting herbicides such as sulfonylureas and imidazolinones13,14. The Zm-Hra gene is regulated by the sorghum ALS promoter8 and potato proteinase inhibitor II (pinII) terminator15. The T-DNA also contains (2) an expression cassette possessing the transformation screenable marker gene ZsGreen. This green fluorescent protein gene ZsGreen from Zoanthus sp. reef coral16 is regulated by a sorghum ubiquitin promoter/intron and rice ubiquitin terminator.
Additionally, the T-DNA contains (3) the morphogenic gene Bbm expression cassette. Bbm is a transcription factor associated with embryo development9,17. Bbm is regulated by the maize phospholipid transferase protein (Pltp) promoter8 and rice T28 terminator18. Zm-Pltp is a gene with strong expression in the embryo scutellar epithelium, silk hairs, and leaf subsidiary cells (flanking the guard cells), low expression in reproductive organs, and no expression in roots8. It also contains (4) the morphogenic gene Wus2 expression cassette. Wus2 is another transcription factor associated with the maintenance of the apical meristem19. Zm-Wus2 is under the control of a maize auxin-inducible promoter (Zm-Axig1)20 and maize In2-1 terminator21. Finally, the T-DNA contains (5) the cre-loxP recombination system. The cre recombinase gene22 is under the control of maize heat shock protein 17.7 (Hsp17.7)23 promoter and potato pinII terminator. Two loxP sites (in the same orientation)24 flank four gene expression cassettes including ZsGreen, cre, Bbm and Wus2.
Because the presence of the morphogenic genes is not desired for plant maturity and subsequent progeny, the heat-induced cre-loxP recombination system was built into the T-DNA to remove morphogenic genes from the maize genome to allow normal callus regeneration and plant development. Upon heat treatment, the expression of CRE protein removes all transgenes except for the Hra selection gene. Successful transformants should be herbicide-resistant but ZsGreen-negative. To further enhance transformation frequency, the Agrobacterium strain also harbors an additional accessory plasmid (PHP71539) that has extra copies of Agrobacterium virulence (vir) genes12.
The QuickCorn method is different from conventional maize transformation protocols, as it does not involve a callus induction step during transformation. During the first week after infection with Agrobacterium, somatic embryos develop on the scutellar epithelium of the infected immature embryos. The embryos are then transferred to a medium with hormones that encourage embryo maturation and shoot formation. Rapidly transferring the somatic embryos onto maturation/shoot formation medium skips the traditional callus stage previously used for maize transformation and permits direct generation of T0 plants8. Compared to previously published maize transformation methods6, the QuickCorn method is faster, more efficient, and less genotype-dependent. Using this method, rooted plants are typically ready to transfer to soil in just 5-7 weeks, rather than the three or more months required by traditional protocols. The purpose of this article is to provide an in-depth description and demonstration of the method, allowing for easier replication in a laboratory setting typically found in most academic institutions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,4-D</td>
			<td>Millipore Sigma</td>
			<td>D7299</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Benzylaminopurine (BAP)</td>
			<td>Millipore Sigma</td>
			<td>B3408</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Millipore Sigma</td>
			<td>D134406</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Millipore Sigma</td>
			<td>A7921</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td></td>
			<td></td>
			<td>To cover the flask</td>
		</tr>
		<tr>
			<td>Ammonium Sulfate</td>
			<td>Millipore Sigma</td>
			<td>A4418</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td></td>
			<td></td>
			<td>To weigh small quantities of chemicals</td>
		</tr>
		<tr>
			<td>Autocalve</td>
			<td>Primus (Omaha, NE)</td>
			<td>PSS5-K</td>
			<td>To autoclave media and tools</td>
		</tr>
		<tr>
			<td>Bacterial culture loop (10 &#181;l)</td>
			<td>Fisher scientific</td>
			<td>22-363-597</td>
			<td>Collects Agrobacterium from plate to transfer to liquid</td>
		</tr>
		<tr>
			<td>Bactoagar</td>
			<td>BD bioscience</td>
			<td>214030</td>
			<td></td>
		</tr>
		<tr>
			<td>Beakers (1 L, 2 L, 4 L)</td>
			<td></td>
			<td></td>
			<td>To mix the chemicals for media</td>
		</tr>
		<tr>
			<td>Benomyl</td>
			<td>Millipore Sigma</td>
			<td>#45339</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach (8.25% Sodium Hypochlorite)</td>
			<td>Clorox</td>
			<td></td>
			<td>For seed sterilization</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Millipore Sigma</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Millipore Sigma</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Millipore Sigma</td>
			<td>C3416</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein Hydrolysate</td>
			<td>Phytotech</td>
			<td>C184</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefotaxime</td>
			<td>Phytotech</td>
			<td>C380</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube (50 mL)</td>
			<td>Fisher scientific</td>
			<td>06-443-19</td>
			<td>Contain liquid medium and Agro suspension</td>
		</tr>
		<tr>
			<td>Cuvette (Semi-micro)</td>
			<td>Fisher scientific</td>
			<td>14955127</td>
			<td>To hold liquid for measuring OD</td>
		</tr>
		<tr>
			<td>Dicamba</td>
			<td>Phytotech</td>
			<td>D159</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital hygrometer</td>
			<td></td>
			<td></td>
			<td>Checking temperature and humidity for heat treatment</td>
		</tr>
		<tr>
			<td>EDTA, Disodium Salt, Dihydrate</td>
			<td>Millipore Sigma</td>
			<td>324503</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube (2.0 mL)</td>
			<td>ThermoFischer Scientific</td>
			<td>AM12475</td>
			<td></td>
		</tr>
		<tr>
			<td>Eriksson&#39;s Vitamins</td>
			<td>Phytotech</td>
			<td>E330</td>
			<td>1000x in liquid</td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td></td>
			<td></td>
			<td>Sterilizing tools and surfaces</td>
		</tr>
		<tr>
			<td>Ferrous Sulfate Heptahydrate</td>
			<td>Millipore Sigma</td>
			<td>F8263</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertilizer, Osmocote Plus 15-9-12</td>
			<td>ICL Specialty Fertilizers (Dublin, OH)</td>
			<td>A903206</td>
			<td>Fertilizer</td>
		</tr>
		<tr>
			<td>Flask (2 L)</td>
			<td>Pyrex</td>
			<td>10-090E</td>
			<td>To autoclave media and tools</td>
		</tr>
		<tr>
			<td>Flats (Standard 1020, open w/holes, 11&#34;W x 21.37&#34;L x 2.44&#34;D)</td>
			<td>Hummert International (Earth City, Mo)</td>
			<td>11300000</td>
			<td>Tray to hold soil and pot insert, fits Humidome</td>
		</tr>
		<tr>
			<td>Forceps (fine-tipped and large)</td>
			<td></td>
			<td></td>
			<td>Fine for handling embryos; larger for large plant materials and use as ear holders</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gold Biotechnologies</td>
			<td>G-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottle (1 L)</td>
			<td>Pyrex</td>
			<td>06-414-1D</td>
			<td>To autoclave medium</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td></td>
			<td></td>
			<td>To adjust volume of media</td>
		</tr>
		<tr>
			<td>Imazapyr</td>
			<td>Millipore Sigma</td>
			<td>37877</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator, 20 &#176;C</td>
			<td>Percival Scientific</td>
			<td>Model I-36NL</td>
			<td>To grow mother plate and incubate embryos during Agro infection</td>
		</tr>
		<tr>
			<td>Incubator, 27 &#176;C</td>
			<td>Percival Scientific</td>
			<td>Model I-36NL</td>
			<td>To grow co-cultivation plate and maize embryo culture</td>
		</tr>
		<tr>
			<td>Incubator, 45 &#176;C</td>
			<td></td>
			<td></td>
			<td>Heat shock treatment</td>
		</tr>
		<tr>
			<td>Insert TO Standard, pots</td>
			<td>Hummert International (Earth City, Mo)</td>
			<td>11030000</td>
			<td>For transplanting plants from rooting to soil, fits flat and Humidome</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td></td>
			<td></td>
			<td>Maintains sterile conditions</td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Phytotech</td>
			<td>P698</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>Millipore Sigma</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>Maize inbred seed B73</td>
			<td>U.S National Plant Germplasm</td>
			<td>id=47638</td>
			<td></td>
		</tr>
		<tr>
			<td>Maize inbred seed Mo17</td>
			<td>U.S National Plant Germplasm</td>
			<td>id=15785</td>
			<td></td>
		</tr>
		<tr>
			<td>Maize inbred seed W22</td>
			<td>U.S National Plant Germplasm</td>
			<td>id=61755</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese Sulfate Monohydrate</td>
			<td>Millipore Sigma</td>
			<td>M7899</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Water purification systems</td>
			<td>Millipore sigma</td>
			<td>MILLIQ</td>
			<td>For tissue culture grade water</td>
		</tr>
		<tr>
			<td>MS Basal Medium</td>
			<td>Millipore Sigma</td>
			<td>M5519</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Basal Salt Mixture</td>
			<td>Millipore Sigma</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr>
			<td>N6 Basal Salt Mixture</td>
			<td>Millipore Sigma</td>
			<td>C1416</td>
			<td></td>
		</tr>
		<tr>
			<td>Paperclips, non-skid</td>
			<td></td>
			<td></td>
			<td>Holding on tassel bags</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>BD bioscience</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (100x15 mm)</td>
			<td>Fisher scientific</td>
			<td>FB0875713</td>
			<td>For bacteria culture medium</td>
		</tr>
		<tr>
			<td>Petri dish (100x25 mm)</td>
			<td>Fisher scientific</td>
			<td>FB0875711</td>
			<td>For the plant tissue culture medium</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Fisher scientific</td>
			<td>AB150</td>
			<td>To adjust pH of media</td>
		</tr>
		<tr>
			<td>Pipette (1 mL)</td>
			<td>ThermoFischer Scientific</td>
			<td>4641100N</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Boxes</td>
			<td>The Container Store</td>
			<td>10048430</td>
			<td>For tissue culture storage and incubation</td>
		</tr>
		<tr>
			<td>Plastic humidy dome (Humi-Dome)</td>
			<td>Hummert International (Earth City, Mo)</td>
			<td>14385100</td>
			<td>Plastic cover for soil flat</td>
		</tr>
		<tr>
			<td>Potassium Iodide</td>
			<td>Millipore Sigma</td>
			<td>793582</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Nitrate</td>
			<td>Millipore Sigma</td>
			<td>P8291</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Millipore Sigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td></td>
			<td></td>
			<td>To weigh chemicals for media</td>
		</tr>
		<tr>
			<td>Scalpel Blade (No. 11, 4 cm)</td>
			<td>Thermo Scientific</td>
			<td>3120030</td>
			<td>remove the top of the kernel crowns for embryo dissection</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td></td>
			<td></td>
			<td>Holding scalpel blades</td>
		</tr>
		<tr>
			<td>Schenk &#38; Hildebrandt Vitamin (S&#38;H vitamin)</td>
			<td>Phytotech</td>
			<td>S826</td>
			<td>100x powder</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td>Cutting ear shoots</td>
		</tr>
		<tr>
			<td>Shoot bag (Canvasback- semi-transparent)</td>
			<td>Seedburo (Des Plaines, IL)</td>
			<td>S26</td>
			<td>Semi-transparent bag to cover ear shoots</td>
		</tr>
		<tr>
			<td>Silver Nitrate</td>
			<td>Millipore Sigma</td>
			<td>S7276</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Molybdate Dihydrate</td>
			<td>Millipore Sigma</td>
			<td>M1651</td>
			<td></td>
		</tr>
		<tr>
			<td>Soiless substrate LC1</td>
			<td>SunGro Horticulture (Agawam, Ma)</td>
			<td>#521</td>
			<td>For growing maize plants</td>
		</tr>
		<tr>
			<td>Spatula (Double Ended Micro-Tapered)</td>
			<td>Fischer Scientific</td>
			<td>2140110</td>
			<td>Dissecting embryos from kernels</td>
		</tr>
		<tr>
			<td>Spatula (with spoon)</td>
			<td>Fisher scientific</td>
			<td>14-375-10</td>
			<td>To measure chemicals for media</td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Millipore Sigma</td>
			<td>S4014</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer (Genesys 10S UV-Vis)</td>
			<td>Thermo Scientific</td>
			<td>840-300000</td>
			<td>Measure OD of Agro suspension</td>
		</tr>
		<tr>
			<td>Stirring bar</td>
			<td>Fisher scientific</td>
			<td>14-513-67</td>
			<td>To mix media</td>
		</tr>
		<tr>
			<td>Stirring hotplates</td>
			<td></td>
			<td></td>
			<td>To mix media</td>
		</tr>
		<tr>
			<td>Syringe (without needle, 60 mL)</td>
			<td>Fisher scientific</td>
			<td>14-823-43</td>
			<td>For filter sterilization</td>
		</tr>
		<tr>
			<td>Syringe filter (0.22 &#181;m)</td>
			<td>Fisher scientific</td>
			<td>09-720-004</td>
			<td>For filter sterilization</td>
		</tr>
		<tr>
			<td>Tassel bag (Canvasback- brown)</td>
			<td>Seedburo (Des Plaines, IL)</td>
			<td>T514</td>
			<td>Bag to cover tassels of non-transgenic plants</td>
		</tr>
		<tr>
			<td>Tassel bag (Canvasback-green stripe)</td>
			<td>Seedburo (Des Plaines, IL)</td>
			<td>T514G</td>
			<td>Bag to cover tassels of transgenic plants</td>
		</tr>
		<tr>
			<td>Thiamine HCl</td>
			<td>Phytotech</td>
			<td>T390</td>
			<td></td>
		</tr>
		<tr>
			<td>Thidiazuron</td>
			<td>Phytotech</td>
			<td>T888</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Millipore Sigma</td>
			<td>T1895</td>
			<td></td>
		</tr>
		<tr>
			<td>Timentin</td>
			<td>Phytotech</td>
			<td>T869</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>Cas #9005-64-5</td>
			<td>surfactant</td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Scientific Industries</td>
			<td>SI0236</td>
			<td>Homogenizes liquids (Agro suspension)</td>
		</tr>
		<tr>
			<td>Water bath (large - Precision model 186)</td>
			<td>Fisher scientific</td>
			<td>any that can fit 4+ 2L flasks and reach 55 &#176;C</td>
			<td>Keeps autoclaved media at optimal temperature</td>
		</tr>
		<tr>
			<td>Weigh dish</td>
			<td>Fisher scientific</td>
			<td>08-732-112</td>
			<td>To measure chemicals for media</td>
		</tr>
		<tr>
			<td>Weighing paper</td>
			<td>Fisher scientific</td>
			<td>09-898-12A</td>
			<td>To measure chemicals for media</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher Scientific</td>
			<td>BP14222</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeatin</td>
			<td>Millipore Sigma</td>
			<td>Z0164</td>
			<td></td>
		</tr>
	</tbody>
</table>

agrobacterium-mediated immature embryo transformation of recalcitrant maize inbred lines using morphogenic genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Demonstrated here is a step-by-step protocol for Agrobacterium-mediated genetic transformation of three public maize inbred lines (B73, Mo17, and W22) that have been significant in the field of maize genetics. Transformation of the three inbred lines could not be achieved using conventional maize transformation protocols5. Figure 1 and Figure 2 show the construct and starting materials, respectively, used here. Ears are generally harvested 9-12 days after pollination. IZEs with lengths ranging between 1.5-2.0 mm are the best explants for transformation for this protocol (Figure 2).
Eight days after infection, ZsGreen-expressing somatic embryos were visualized under the GFP channel of a fluorescent microscope (Figure 3). Infected IZEs were subjected to heat treatment 8 days after infection (steps 5.1 and 5.2). This treatment induced the expression of CRE recombinase that excised the Bbm, Wus2, cre, and ZsGreen expression cassettes flanked between the two loxP sites (Figure 1). The heat-treated tissues were then cultured on shoot formation medium containing the herbicide imazapyr for selection of transformed tissue after morphogenic gene removal.
Proliferating tissues with maturing embryos or shoot buds that were resistant to imazapyr were observed around 3-4 weeks after infection (Figure 4). Some imazapyr-resistant tissues were negative for ZsGreen, suggesting that cre-mediated excision likely occurred in these tissues (Figure 4). After the tissues were moved to rooting medium and light incubation, shoots started to develop (Figure 5). Healthy and vigorous growing shoots with well-developed roots were harvested (Figure 5). Some tissues appeared to have multiple shoots (Figure 5E,F,G). This type of &#34;grassy&#34; regenerant may be due to clonal plants having identical transgene integration patterns. Molecular biological analysis is required to genotype these plants.
All three public inbred lines responded well using this protocol as well as the construct used in this work. W22 produced the highest frequency of imazapyr-resistant shoots, with a frequency of approximately 14% (about 14 transgenic shoots per 100 infected immature embryos). Both B73 and Mo17 produced about 4% transgenic shoots. These frequencies indicate all transgenic shoots, including both plants carrying the morphogenic genes and plants with the morphogenic gene removed by the CRE-mediated excision.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60782/60782fig1.jpg" /> 
Figure 1: Schematic representation of the T-DNA region of the binary plasmid PHP81430. RB = right T-DNA border; loxP = CRE recombinase target site; Axig1pro:Wus2 = maize auxin-inducible promoter (Zm-Axig1) + Zm-Wus2 + maize In2-1 terminator; Pltppro:Zm-Bbm = maize phospholipid transferase protein (Zm-Pltp) promoter + Zm-Bbm + rice T28 terminator (Os-T28); Hsppro:cre = maize heat shock protein 17.7 promoter (Zm-Hsp17.7) + cre recombinase gene + potato proteinase inhibitor II (pinII) terminator; Ubipro:ZsGreen = sorghum ubiquitin promoter/intron (Sb-Ubi) + green fluorescent protein ZsGreen gene + rice ubiquitin terminator (Os-Ubi); Hra cassette = sorghum acetolactase synthase (Sb-Als) promoter + maize Hra (Zm-Hra) gene + pinII terminator; LB = left T-DNA border; colE1, replication origin of plasmid ColE125; SpecR = spectinomycin resistant gene aadA1 from Tn21 for bacterium selection26; Rep A,B,C = replication origin from pRiA4 of Agrobacterium rhizogenes27. <a href="https://www.jove.com/files/ftp_upload/60782/60782fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60782/60782fig2c.jpg" /> 
Figure 2: Starting materials. B73 ears harvested 12 days post-pollination (A). Immature embryos of B73 (B), Mo17 (C), and W22 (D). <a href="https://www.jove.com/files/ftp_upload/60782/60782fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60782/60782fig3c.jpg" /> 
Figure 3: Tissue development on resting medium 1 week post-infection. Embryos (8 days post-infection) under a florescence microscope (GFP filter) showing GFP expressing somatic embryos of Mo17 (A) and W22 (B). Developing tissue (B73) under bright-field (C) and GFP filter (D). <a href="https://www.jove.com/files/ftp_upload/60782/60782fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60782/60782fig4c.jpg" /> 
Figure 4: Tissue development on maturation medium with selection. A W22 maturation plate (A). Developing tissue (Mo17, 15 days post-infection) under bright-field (B) and GFP filter (C). Developing tissue (Mo17, 28 days post-infection) under bright-field (D) and GFP filter (E). Arrows point to regenerating tissues that are lacking GFP expression, suggesting the excision of ZsGreen gene between the loxP sites after heat-induced CRE protein activity. <a href="https://www.jove.com/files/ftp_upload/60782/60782fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60782/60782fig5c.jpg" /> 
Figure 5: Tissue development on rooting media. Shoots of W22 (A), B73 (B), and Mo17 (C,D). Event with multiple shoots (grassy regenerants) of B73 (E) and W22 (F,G). Shoots with roots of B73 (H) and W22 (I). <a href="https://www.jove.com/files/ftp_upload/60782/60782fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Media compositions for maize transformation. <a href="https://www.jove.com/files/ftp_upload/60782/table1.xlsx" target="_blank">Please click here to view this table (Right click to download).</a>

Watch this Scientific Journal Video about Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes at JoVE.com

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

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

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

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

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22.5K Views.  Iowa State University. Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

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

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

Visualization of Cellular Interactions Between the Maize Sheaths and Fungi

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 &#181;L drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 &#176;C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

Author Spotlight: Investigating Fungal Pathogenicity Mechanisms in Maize 

This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...

In Planta Transient Transformation of Cacao Plants Using Agrobacterium tumefaciens

Vacuum-Forced Agroinfiltration for In planta Transformation of Recalcitrant Plants: Cacao as a Case Study

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.

Author Spotlight: Tissue Culture-Free Agrobacterium-Mediated Transformation in Large Woody Plants

Here, we present the first protocol for localized vacuum infiltration for in vivo studies of the genetic transformation of large-sized plants. Using this methodology, we achieved for the first time the Agrobacterium-mediated in planta transient transformation of cacao.

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation ...

Agrobacterium-Mediated Hairy Root Induction and Regeneration in Plants

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

Author Spotlight: Optimizing Hairy Root-Based Transformation Protocols for Enhanced Efficiency in Brassicaceae

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.