Protocol of Agrobacterium-Mediated Transformation for Rice

Main Reagents

GUS staining solution: 100 mmol/L NaPO4 (pH 7.0); 0.1% Triton X-100; 10 mmol/L EDTA; 0.5 mmol/L potassium ferricyanide; cefotaxime, ethanol, sodium hypochlorite solution.

Main Equipment

High-speed centrifuge, incubator, artificial climate chamber.

Experimental Materials

Rice seeds.

Experimental Steps

1. Induction of young embryonic healing tissue

The seeds were taken 12-15 days after flowering, disinfected with 70% ethanol for 1 minute and rinsed 2-3 times with sterile water; then disinfected with 2% effective chlorine sodium hypochlorite solution by shaking for more than 60 minutes and rinsed 4-5 times with sterile water, then the young embryos were isolated under aseptic conditions, connected to N₆D₂ medium and cultured at 28°C for 4 days in the dark.

2. Culture of Agrobacterium tumefaciens

The single clone of Agrobacterium tumefaciens was picked from YEB solid medium and inoculated into 20 ml of YEB liquid medium containing Km 50 mg /l and incubated at 28°C until late logarithmic growth; then 0.5 mI was transferred to 50 mI of the same YEB medium and incubated under the same conditions until OD600 was about 0.5.

3. Co-culture and transformation, screening, differentiation

(1)The cultured Agrobacterium rhizogenes was centrifuged at 4000 g for 10 min and the precipitate was resuspended with an equal volume of AAM-AS medium.

(2) Healing tissues derived from young embryos of C. intermedia 11, which were precultured for 4 d, were invaded into this AAM-AS solution for 20 min, blotted with sterile filter paper and transferred to N6D2C medium (with a sterile filter paper on the surface of the medium) at 25°C and incubated in the dark for 3 d.

(3) The healing tissue was washed 4-5 times with sterile water containing 300 mg/L cephalexin, blotted dry on sterile filter paper and transferred to N6D2S1 medium to screen the generation.

(4) After two weeks, transfer to N6D2S2 medium to screen the second generation (2 weeks/generation).

(5) Resistant healing tissues that were screened for vigorous growth after 3 generations were removed, transferred to predifferentiation medium and cultured in a differentiation incubator (12-hour photoperiod, 28°C during the day and 25°C at night) for 7 d; they were then transferred to differentiation medium and cultured in a differentiation incubator until regeneration seedlings were produced.

(6) Regenerated plants rooted and strengthened on rooting medium.

(7) When the seedlings grow to about 10 cm, open the container sealing film, refine the seedlings for 2-3 d, and then move the seedlings into the artificial climate chamber for cultivation.

4. Media used in rice tissue culture and transformation process and its composition

N₆D₂ medium	N6 salts and vitamins, 500 g/l of hydrolyzed casein, 30 g/l of sucrose, 2 mg/l of 2,4-D, 2.5g/l gelrite.
N₆D₂S₁ medium	N₆D₂, 25 mg/l hygromycins, 600 mg/l cefotaxime, pH5.8.
N₆D₂S₂ medium	N₆D₂, 50 mg/l hygromycins, 300 mg/l cefotaxime, pH5.8.
AAM-AS medium	AA sole i aminokwasy, witaminy MS,100 μmol/l acetosyringone, pH5.2.
N₆D₂C	N₆D₂, 10 g/l glucose, 100 μmol/l acetosyringone, pH5.2.
Differentiation medium	MS salts and vitamins, 300 g/l hydrolyzed casein, 50 mg/l hygromycins, 3 mg/l 6-BA,2.5 mg/l KT, 0.2 mg/l ZT, 2.5 g/l gelrite, pH5.8.
Rooting medium	1/4 MS salts, MS vitamins, 1 mg/l dmethoate, 0.5 mg/l NAA, 6.5 g/l agar powder, pH5.8.

5. Histochemical analysis of transformed plants

The resistant healing tissues produced after transformation by Agrobacterium tumefaciens and the leaf and root segments of the transformed plants were put into GUS staining solution, pumped for a few minutes, and then placed at 37°C for overnight warming to observe the blue reaction. The stained tissues were decolorized with 70% ethanol.

* For research use only. Not intended for any clinical use.

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Protocol of Agrobacterium-Mediated Transformation for Rice

Main Reagents

Main Equipment

Experimental Materials

Experimental Steps

1. Induction of young embryonic healing tissue

2. Culture of Agrobacterium tumefaciens

3. Co-culture and transformation, screening, differentiation

4. Media used in rice tissue culture and transformation process and its composition

5. Histochemical analysis of transformed plants

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