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

Most maize inbred lines cannot be genetically transformed using conventional transformation protocols. Here, we describe a QuickCorn transformation method that is fast and less genotype-dependent. The QuickCorn method utilizes maize transcription factors BABY BOOM and WUSCHEL.

When incorporated in the transformation vector system, these genes work synergistically to stimulate embryogenic growth. Unlike conventional maize transformation protocols, the QuickCorn method does not involve a callus induction step during transformation. The T-DNA region of the binary vector used in our work contains three key components, morphogenic genes, marker genes, and the cre/loxP recombination system.

The heat-induced cre/loxP recombination system was included in the T-DNA to remove the morphogenic genes from the maize genome to allow normal callus regeneration in plant development. About two to three days after silks have emerged and if pollen will be available the following day, cut the silks and husk with 70%ethanol-sterilized scissors, roughly 2 1/2 centimeters below the end of the husk leaves, where the silks emerge, and cover the silk with a shoot bag. Once anthers emerge from a tassel, cover the tassel with a tassel bag, and place a non-skid paper clip at the base of the bag around the stalk.

The morning after placing the tassel bag, gently bend the plant, and tap the bag to encourage pollen to be released. Then remove the tassel bag, and fold the top of the bag over to prevent pollen from escaping. To pollinate a recipient plant, expose the silks, and quickly pour pollen from the tassel bag onto the silks.

Immediately cover the pollinated ear with the tassel bag, and secure the base of the bag around the stalk with staples. To check the immature embryo size, nine to 12 days after pollination, gently pull down the husk to expose the kernels at about 1/3 to 1/4 of the circumference of the ear and about 1/3 of the distance down the ear. Use a scalpel to slice off the cap of a single kernel that appears similar to the majority of other kernels in size and color, and use a spatula with a ruler to extract the embryo.

Then use the ruler or a caliper to measure the length of the embryo. Within one to four days of harvest, remove the husks and silks from the harvested ears, and insert an appropriate handle into the top or the base of each ear. Submerge the ears in a large container of disinfection bleach solution in a sterile laminar flow hood with the handle facing up.

After 20 minutes, rinse the ears three times with a generous volume of fresh sterile distilled water for five minutes per wash before allowing the ears to dry for several minutes. Next, fill one two-milliliter microcentrifuge tube per ear with 700A liquid medium, and use a sterile scalpel to remove the top one to two millimeters of each kernel crown to expose the endosperm of the ear. Locate the immature embryo within the kernel on the side facing the tip of the ear, near the attachment to the cob.

For top handler and right-handed operators, rest the ear on a large sterile Petri dish, and insert a micro spatula into the endosperm in the pericarp furthest away from the embryo. Gently twist upward to dislodge the endosperm and to expose the embryo, and use the spatula to carefully place the embryo into one tube of 700A liquid medium. For base handler operators holding the ear with the left hand, insert a micro spatula into the endosperm in the pericarp furthest away from the embryo, and gently twist upward to dislodge the endosperm.

To culture the embryos in an Agrobacterium suspension culture, collect bacteria from a freshly prepared working plate into 10 milliliters of 700A liquid medium, and vortex to suspend the bacteria culture completely. Measure the optical density at a wavelength of 550 nanometers, and wash the embryos with one milliliter of fresh 700A medium. Immerse the embryos in one milliliter of Agrobacterium suspension, and vortex on a low setting for 30 seconds.

Settle the embryos on the bench with the tubes in a horizontal orientation for five minutes before transferring the entire contents of each tube onto individual plates of 562V co-cultivation medium. Gently swirl the plates to distribute the embryos evenly, and aspirate the excess Agrobacterium suspension. Carefully orient the embryos with the dome-shaped side facing up, taking care to avoid damaging the embryos.

Then place plates into plastic boxes for an overnight incubation at 21 degrees Celsius in the dark. The next morning, carefully transfer infected embryos onto resting medium 605T, place about 30 embryos per plate, scutellum side up, for a four-to 10-day incubation at 26 degrees Celsius in the dark. At around seven days, somatic embryo development can be observed on the surface of the zygotic scutellum.

At the end of the resting period, place the box of embryos in a 45-degree Celsius incubator with 70%relative humidity for two hours, followed by a one-to two-hour incubation at 26 degrees Celsius in the dark. At the end of the incubation, place 10 to 15 heat-shocked immature embryos onto individual plates of shoot formation medium supplemented with 05 milligrams per liter of the herbicide imazapyr as a selective agent. Carefully remove any coleoptiles as needed, and return the embryos to the 26-degree Celsius dark incubator for two weeks.

At the end of the incubation, transfer about eight pieces of tissue per plate onto rooting medium plates for a one-to two-week incubation with 16 hours of light and eight hours of dark at 27 degrees Celsius. As plantlets develop, place one stronger plantlet containing both shoots and vigorous roots onto individual plates of rooting medium under light for another seven to 14 days. Allow shoots that are not fully developed to be incubated on the same medium for another one to two weeks until they are ready to be moved to soil.

As the plant becomes more vigorous, rinse the roots with tap water to remove agar. Then transplant the individual plants into three-inch pots containing a pre-wetted soilless substrate in a tray with drain holes, and place the tray in a growth chamber with or without a plastic humidity dome. Nine to 14 days after pot transfer, transplant each plantlet with soil into a 1.5-gallon pot.

Add a controlled-release fertilizer to the pot, and maintain the plants in the greenhouse. When ear shoots begin to emerge from the plant, use a semitransparent shoot bag to cover the shoots so that the emerging silks can be observed without removing the bag. Then pollinate the plants at the appropriate stage of development as demonstrated.

The maize ears are generally harvested nine to 12 days after pollination. Immature embryos with lengths ranging between 1.5 and two millimeters are the best explants for transformation for this protocol. Eight days after infection, ZsGreen-expressing somatic embryos can be visualized by fluorescence microscopy.

Heat treatment eight days after infection induces cre recombinase expression, resulting in the excision of the morphogenic gene, cre, and ZsGreen expression cassettes flanked between the two loxP sites. After three to four weeks of culture on shoot formation medium containing herbicide, proliferating tissues with maturing embryos or shoot buds resistant to the herbicide can be observed. Some of the herbicide-resistant tissues may be negative for ZsGreen, suggesting that cre-mediated excision likely occurred in these tissues.

After moving the tissues to rooting medium and light incubation, healthy, vigorous, growing shoots with well-developed roots can be harvested. Note that some tissues may appear to have multiple shoots possibly due to clonal plants having identical transgene integration patterns. The QuickCorn method can greatly improve maize transformation efficiency and expand the list of transformable genotypes.

The protocol can be successfully reproduced by researchers with minimum maize transformation training. Using the QuickCorn method, rooted plants should be ready to transfer to soil in just five to seven weeks after the day of the infection. Pay attention to the medium composition, the timing of the subcultures, and the temperature and lighting conditions.

The quality of the starting materials is also essential for successful transformation. The chemicals, bleach solution, and herbicide used in this protocol are biohazardous. Please make sure to wear the appropriate personal protection equipment during their use.