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

Young inflorescences as explant for callus production are important for establishing a rapid and efficient gene transformation and a regeneration method for revealing gene function as well as for crop improvement. Young inflorescences as explant for callus production are important for establishing a rapid and efficient gene transformation and the regeneration method for revealing gene function. Video demonstration of this technique will help the viewer understand the process of maintaining the materials to produce that desired transgenic align without wasting time and effort.

Begin by collecting young rice inflorescences from the paddy field or a greenhouse at meiosis stage, making sure that they are covered with the leaf sheath. Wipe each inflorescence with a 70%alcohol swab and let it dry before cutting. Bring the inflorescence to a sterile lab bench and cut it into small pieces with sterilized scissors, then transfer the cuttings to a Petri plate containing NBD2 medium.

Incubate the plate in the dark at 26 degrees Celsius for 10 to 14 days to induce callus. To perform transformation, transfer a single colony from the white EB plate with selective antibiotics to five milliliters of liquid YEB medium containing the same antibiotics in a 50 milliliter conical sterile test tube. Shake the tube on an orbital shaker at 250 times g and 25 to 28 degrees Celsius until bacteria grow to an OD 600 of 0.5.

Add one milliliter of bacterial suspension to 100 milliliters of YEB medium with the same selective antibiotics in a 250 milliliter conical flask and shake the flask on an orbital shaker at 250 times g and 28 degrees Celsius for four hours. Centrifuge the culture at 4, 000 g for 10 minutes at room temperature to collect the bacteria. Discard the supernatant and resuspend the pellet with AAM AS medium, then dilute the suspension to an OD 600 of 0.4.

After the incubation, collect around 150 healthy light yellow embryogenic calli into a 150 milliliter sterile flask. Add 50 to 75 milliliters of the bacterial cell suspension into the flask and then add 10 to 25 milliliters of fresh AAM AS medium to immerse the calli for 10 to 20 minutes, shaking occasionally. Pour the bacterial suspension out of the flask carefully and dry the calli with sterile filter paper, then place them on a Petri dish with NBD AS medium and cover them with filter paper.

Incubate the calli at 25 to 28 degrees Celsius in the dark for three days, checking them for bacterial overgrowth. After three days of co-cultivation, transfer the calli to a sterile Petri dish using filter paper, then air dry them for two hours on a clean bench. Ensure that the calli are not adhered to the filter paper and transfer them to the primary selection medium NBD2.

After two weeks, transfer the calli evenly to a new plate containing fresh selection medium, then move the calli to fresh MS medium for differentiation and the shoot buds to MS medium with 10 milligrams per liter Hygromycin to proliferate more shoots. Transfer the new shoots into half MS medium for root induction and culture them under light at 25 to 28 degrees Celsius. The transformed plants should produce roots within two weeks.

To perform reproductive phenotype observation, remove the palea and lemma from the florid and image the whole anthers under a stereo microscope. To observe pollen viability, pick mature rice spikelets before the flower anthesis and remove the palea and lemma to release the anthers. Take six anthers and place them on a glass slide.

Add one drop of distilled water, crush the anther with tweezers to release the pollen grains, then add two to three drops of iodine solution and cover with a coverslip. Observe the slide under a low magnification microscope. Pollen grains dyed black show more vigorous viability while grains with no color or those dyed yellow brown are stunted or degenerated.

To perform transverse sectioning microscopy, use 0.5%toluidine blue solution to stain the slides for 30 minutes, then rinse them with water and dry them in the fume hood. Once dry, seal the slides with neutral tree glue, gently place a coverslip on the slide and dry them in the fume hood, then image the sample under a microscope. This protocol was used to create a male sterile line by Agrobacterium-mediated genetic transformation in rice.

The embryogenic calli were directly used for the transformation or subcultured for proliferation to obtain more calli for further infection. After co-cultivating for 48 hours, the calli were shifted to a second round of selection medium. After 10 to 14 days, transformed calli showed newly formed microcalli while the untransformed calli turned brown and died.

Later, healthy and creamy colored calli were transferred into regeneration medium. The transformed calli gradually showed green spots. These green spots were cultured in regeneration medium to allow for shoot growth and then shifted into half MS medium to induce roots.

Later, healthy and vigorous growing shoots with well-developed roots were harvested. In total, 21 regenerated seedlings were obtained. PCR amplification for the Hygromycin region showed that the transformation frequency was approximately 85.71%The transformants were genotyped to identify the mutations at the sgRNA target sites.

Among the 18 transgenic plants, eight heterozygous lines and three homozygous lines were CRISPR CAS9 positive. The homozygous knockout mutants were validated by basic male reproductive organ observation. The anthers of osabcg15 were smaller and paler than those of the wild type and lacked mature pollen grains.

Transverse sectioning microscopy was performed to investigate the anther morphological defects in osabcg15. At stage 10, wild-type microspores became round shaped and vacuolated with dark blue stained exine. In contrast, the osabcg15 microspores collapsed and degraded.

When attempting this protocol, it is important to ensure that the collected material is in meiosis stage and to control the air dry time of the calli.