Agrobacterium-Mediated Genetic Transformation: A Method to Genetically Transform the Rice Genome via Genetically Engineered Agrobacterium tumefaciens

Overview

In this video, we demonstrate genetic transformation of the rice genome using an Agrobacterium tumefaciens binary vector. This bacterium facilitates the transfer of a foreign DNA plasmid using Agrobacterium virulence proteins.

Protocol

1. Rice genetic transformation and plant tissue culture

1. Callus induction and regeneration
   NOTE: Use fresh young rice inflorescences as the starting material to induce callus (Figure 1).
   1. Collect the young inflorescences from the paddy field or green house at meiosis stage, determined by floret length of 1.6-4.8 mm (Figure 1A). Ensure that the rice inflorescences are covered with leaf sheath. Wipe each inflorescence with a 70% alcohol swab and let it dry before cutting.
   2. Bring the inflorescence to a clean sterile bench. Cut it into small pieces (the smaller the better) with sterilized scissors and then transfer cuttings to a Petri plate containing NBD2 medium (Figure 1B, Table 1).
   3. Incubate the plate in the dark at 26 °C for about 10-14 days to induce callus (Figure 2A).
      NOTE: Use the newly formed callus for Agrobacterium infiltration (step 1.2.7) directly or recondition one more time in fresh NBD2 medium to obtain more callus. Transfer the callus into the new NBD2 medium every 8–10 days.
2. Transformation and co-cultivation
   1. Use A. tumefaciens bacteria containing the binary plasmid with sgRNA-CAS9 construct for transformation. Store the A. tumefaciens strains in YEB medium (Table 1) with 50% glycerol at -80 °C for further use.
   2. Streak A. tumefaciens from a -80 °C glycerol stock to YEB agar medium containing selective antibiotics (Table 1) and grow at 25–28 ˚C for 48–72 h, to allow colonies to appear.
   3. Inoculate a single colony from the YEB plate with selective antibiotics to 5 mL of liquid YEB medium containing the same selective antibiotics (Table 1), in a 50 mL conical sterile test tube. Shake on an orbital shaker at 250 x g, at 25–28 °C until bacteria grow to an OD₆₀₀ of 0.5.
   4. Add 1 mL of bacterial suspension to 100 mL of YEB medium (Table 1) with the same selective antibiotics in a 250 mL conical flask and shake on an orbital shaker at 250 x g, at 28 °C for 4 h.
   5. Centrifuge at 4,000 x g for 10 min at room temperature to collect bacteria. Discard the supernatant.
   6. Resuspend the bacterial pellet with AAM-AS medium (Table 1) and dilute the suspension to an OD₆₀₀ = 0.4.    
      NOTE: The perfect OD of bacterial suspension is critical for efficient transformation. It helps in eliminating excess bacterial growth on the callus.
   7. Collect around 150 healthy growing light-yellow fragile embryogenic calli from step 1.1.3 into a 150 mL sterile flask. Add 50–75 mL bacterial cell suspension from step 1.2.6 into the sterile flask, and then add some 10–25 mL fresh AAM-AS medium to immerse the calli for 10–20 min, shaking occasionally.
   8. Pour out the bacterial suspension from the flask carefully and dry the excess bacterial suspension from the callus using sterile filter paper #1 or tissue paper. Then place them on a Petri dish with NBD-AS medium (Table 1) covered with a sterile filter paper #1. Incubate at 25–28 °C in the dark for 3 days and check for bacterial overgrowth.
3. Selection for resistant callus
   1. After 3 days of co-cultivation, transfer the calli to a sterile Petri dish with a sterile filter paper.
   2. Air-dry the calli for 2 h on a clean bench. Ensure that the callus is not adhered to the filter paper.
   3. Transfer the calli evenly using sterile tweezer to the primary selection medium NBD2 (with 40 mg/L hygromycin and 250 mg/L timentin) (Table 1). Culture calli for 2 weeks at 25–28 °C in the dark.
   4. After 2 weeks, transfer calli evenly to a new plate containing fresh selection medium NBD2 (with 40 mg/L hygromycin and 250 mg/L timentin) (Table 1). Culture the calli for another 2 weeks at 25–28 °C in the dark.
4. Callus differentiation
   1. Transfer callus to fresh MS Medium (with 25 mg/L hygromycin and 150 mg/L timentin) (Table 1). Culture under the light at 25–28 °C for around 2 weeks.
   2. Repeat step 1.4.1 one more time.
   3. Transfer the shoot buds to the new MS medium (with 10 mg/L hygromycin) to proliferate more shoots.

Table 1: Media compositions for rice transformation. Please click here to download this table.

Representative Results

Figure 1: Starting materials for inducing callus. (A) Young rice inflorescences at meiosis stage. (B) Dissected young inflorescences growing on the NBD2 medium. Bar = 2 cm in (A).

Figure 2: Procedure for producing transgenic lines by Agrobacterium-mediated transformation. (A) Rice calli generated from the young inflorescences. (B) Subculture of calli. (C) Incubation of calli with A. tumefaciens. (D) Co-cultivation of calli with A. tumefaciens. (E) First selection cycle of transformed calli in the presence of Hygromycin (40 mg/L). (F) Second selection cycle of transformed calli in the presence of Hygromycin (40 mg/L). (G) First regeneration of transformed calli. (H) The second regeneration of transformed calli, noting the green shoot point in the culture plate. (I) Shoot induction culture. (J) Root induction.