Team:SCAU-China/Protocol
Protocol
Vector construction protocol
Transformation of rice
1.Brief on the procedure:
Callus induction→ Subculture of callus → Co-culture of callus with Agrobacterium → Selection of resistant callus→ Differentiation of resistant callus → Rooting the seedling → Hardening the seedling →Transplanting the seedling.
2.Detailed steps
2.1 Callus induction
2.1.1 Medium Preparation
| Component | Dosage |
|---|---|
| 10×N6 macroelement solution | 50 mL |
| 1000×B5 microelement solution | 0.5 mL |
| 100×B5 vitamin solution | 5 mL |
| 100×Ferric salt solution | 5 mL |
| 0.5 mg/mL 2,4-D | 3 mL |
| Casein enzymatic hydrolysate | 150 mg |
| L-Proline | 250 mg |
| L-Glutamine | 250 mg |
| Sucrose | 15 g |
| Add distilled water until the medium volume reach 500 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.8 with 1 mol/L NaOH | |
| Phytagel | 1.5 g |
2.1.2 Experimental procedures
(1)Put 400 rice seeds into a 50 mL Erlenmeyer flask.
(2)Prepare 75% ethanol solution.
(3)Pour some 75% ethanol solution into the Erlenmeyer flask which contain the seeds. Keep shaking the Erlenmeyer flask in 1 minute at most.
(4)Decant the 75% ethanol from the Erlenmeyer flask.
(5)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water. Repeat this step for 5 times.
(6)Prepare 1.5% sodium hypochlorite solution.
(7)Pour some 1.5% sodium hypochlorite solution into the Erlenmeyer flask and seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker for 30 minutes.
(8)Decant the 1.5% sodium hypochlorite solution.
(9)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water, Repeat this step for 5 times.
(10)Repeat step 6 to 9.
(11)Pick out seeds by a sterile tweezer and put seeds on a sterile filter paper.
(12)Place seeds in the laminar flow cabinet and dried by blowing for about 2 hours.
(13)Put sterile seeds on the surface of the callus initiation medium and seal plates with parafilm.
(14)Incubate seeds in the dark for about 14 days.
2.2 Subculture of callus
2.2.1 Medium preparation
| Component | Dosage |
|---|---|
| 10×N6macroelement solution | 50 mL |
| 1000×B5microelement solution | 0.5 mL |
| 100×B5vitamin solution | 5 mL |
| 100×Ferric salt solution | 5 mL |
| 0.5 mg/mL 2,4-D | 3 mL |
| Casein enzymatic hydrolysate | 150 mg |
| L-Proline | 250 mg |
| L-Glutamine | 250 mg |
| Sucrose | 15 g |
| Add distilled water until the medium volume reach 500 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.8 with 1 mol/L NaOH | |
| Phytagel | 1.5 g（3 g/L） |
2.2.2 Experimental procedures
(1)Place the callus initiation medium in the laminar flow cabinet.
(2)Use a sterile tweezer to divide callus from seeds and put callus on the subculture medium. Seal plates with parafilm.
(3)Incubate callus in the dark for 5 days.
2.3 Co-culture of callus with Agrobacterium.
2.3.1 Medium preparation
(1)
| Component | Dosage |
|---|---|
| 10×MSmacroelement solution | 25 mL |
| 1000×B5microelement solution | 0.25 mL |
| 100×B5 vitamin solution | 2.5 mL |
| 100×Ferric salt solution | 2.5 mL |
| 0.5 mg/mL 2,4-D | 1.5 mL |
| Casein enzymatic hydrolysate | 125 g |
| Inositol | 0.5 g |
| Sucrose | 7.5 g |
| Glucose | 7.5 g |
| Add distilled water until medium volume reach 250 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.2 with 1 mol/L NaOH | |
| 1000×Acetosyringone (Add before using the medium) |
0.25 mL |
(2)
| Component | Dosage |
|---|---|
| 10×MSmacroelement solution | 25 mL |
| 1000×B5microelement solution | 0.25 mL |
| 100×B5 vitamin solution | 2.5 mL |
| 100×Ferric salt solution | 2.5 mL |
| 0.5 mg/mL 2,4-D | 1.5 mL |
| Casein enzymatic hydrolysate | 125 g |
| Inositol | 0.5 g |
| Sucrose | 7.5 g |
| Glucose | 7.5 g |
| Add distilled water until medium volume reach 250 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.3 with 1 mol/L NaOH | |
| Agar | 3.5 g |
| 1000×Acetosyringone (Add before dividingthe medium into plates) |
0.25 mL |
2.3.2 Experimental procedures
(1)Place the subculture medium into the laminar flow cabinet.
(2)Pick out the callus and put them on a sterile filter paper. Dry the surface of callus by blowing.
(3)Add 0.25 mL Acetosyringone to co-culture liquid medium.
(4)Pour about 35 mL co-culture liquid medium into a 50 mL Erlenmeyer flask.
(5)Use a sterile stainless steel spoon to collect Agrobacterium transforment strain and suspend in the co-culture liquid medium. The co-culture liquid medium contain Agrobacterium also called Agrobacterium suspension.
(6)Adjust the OD600 of Agrobacterium suspension to 0.1.
(7)Seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker in 27 ℃ for 30 minutes.
(8)After 30 minutes, take the Erlenmeyer flask out and put it in the laminar flow cabinet. Pick callus into the Agrobacterium suspension and gentle shake the Erlenmeyer flask for 20 minutes. Ensure that each callus contact with the Agrobacterium suspension directly.
(9)Decant the Agrobacterium suspension. Pick out callus and put them on a sterile filter paper.
Dry the surface of callus by blowing.
Dry the surface of callus by blowing.
(10)At the same time, put a sterile filter paper on the surface of the co-culture medium. Dry the surface of the co-culture medium by blowing.
(11)When the surface of callus become dry, put them on the surface of the filter paper above the co-culture medium.
(12)Seal plates with parafilm. Incubate in the dark at 25 ℃ for 3 days.
2.4 First time selection of resistant callus
2.4.1 Medium preparation
| Component | Dosage |
|---|---|
| 10×N6macroelement solution | 50 mL |
| 1000×B5 microelement solution | 0.5 mL |
| 100×B5 vitamin solution | 5 mL |
| 100×Ferric salt solution | 5 mL |
| 0.5 mg/mL 2,4-D | 3 mL |
| Casein enzymatic hydrolysate | 150 mg |
| L-Proline | 250 mg |
| L-Glutamine | 250 mg |
| Sucrose | 15 g |
| Add distilled water until the medium volume reach 500 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.8 with 1 mol/L NaOH | |
| Phytagel | 3 g |
| 1000×Hygromycin B (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Cefazolin sodium (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Carbenicillin sodium (Add before dividing the medium into plates) |
0.5 mL |
2.4.2 Experimental procedures
(1)Place the co-culture medium in the laminar flow cabinet.
(2)Add 1 mL 1000×Cefazolin sodium and 1 mL 1000×Carbenicillin sodium to 1 L sterile distilled water. This solution is Agrobacterium eluent.
(3)Pick out callus and put them in a 50 mL Erlenmeyer flask.
(4)Add about 35 mL Agrobacterium eluent to the Erlenmeyer flask. Shake the Erlenmeyer flask for one minute. Decant the Agrobacterium eluent.
(5)Repeat step 4 for about 20 times. Ensure that most of the Agrobacterium on callus are eluted.
(6)Pick out callus and put them on a sterile filter paper. Dry the surface of callus by blowing.
(7)Put callus on the selecting medium.
(8)Seal plates with parafilm.
(9)Culture in the dark for 30 days.
2.5 Second time selection of resistant callus
2.5.1 Medium preparation
| Component | Dosage |
|---|---|
| 10×N6 macroelement solution | 50 mL |
| 1000×B5 microelement solution | 0.5 mL |
| 100×B5 vitamin solution | 5 mL |
| 100×Ferric salt solution | 5 mL |
| 0.5 mg/mL 2,4-D | 3 mL |
| Casein enzymatic hydrolysate | 150 mg |
| L-Proline | 250 mg |
| L-Glutamine | 250 mg |
| Sucrose | 15 g |
| Add distilled water until the medium volume reach 500 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.8 with 1 mol/L NaOH | |
| Phytagel | 3 g |
| 1000×Hygromycin B (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Cefazolin sodium (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Carbenicillin sodium (Add before dividing the medium into plates) |
0.5 mL |
2.5.2 Experimental procedures
(1)Place the first time selecting medium in the laminar flow cabinet.
(2)Pick out callus and put them on the second time selecting medium.
(3)Seal plates with parafilm.
(4)Culture in the dark for 30 days.
2.6 Differentiation of resistant callus
2.6.1 Medium preparation
| Component | Dosage |
|---|---|
| 10×N6 macroelement solution | 50 mL |
| 1000×MS microelement solution | 0.5 mL |
| 100×B5 vitamin solution | 5 mL |
| 100×Ferric salt solution | 5 mL |
| 0.5 mg/mL 6-B,A | 3 mL |
| 0.1 mg/mL NAA | 5 mL |
| Sorbitil | 9.1 g |
| Sucrose | 10 g |
| Add distilled water until the medium volume reach 500 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.8 with 1 mol/L NaOH | |
| Phytagel | 2 g |
| 1000×Hygromycin B (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Cefazolin sodium (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Carbenicillin sodium (Add before dividing the medium into plates) |
0.5 mL |
2.5.2 Experimental procedures
(1)Place the first time selecting medium in the laminar flow cabinet.
(2)Pick out callus and put them on the second time selecting medium.
(3)Seal plates with parafilm.
(4)Culture in the dark for 30 days.
2.6 Differentiation of resistant callus
2.6.1 Medium preparation
| Component | Dosage |
|---|---|
| 10×N6 macroelement solution | 50 mL |
| 1000×MS microelement solution | 0.5 mL |
| 100×B5 vitamin solution | 5 mL |
| 100×Ferric salt solution | 5 mL |
| 0.5 mg/mL 6-B,A | 3 mL |
| 0.1 mg/mL NAA | 5 mL |
| Sorbitil | 9.1 g |
| Sucrose | 10 g |
| Add distilled water until the medium volume reach 500 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.8 with 1 mol/L NaOH | |
| Phytagel | 2 g |
| 1000×Hygromycin B (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Cefazolin sodium (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Carbenicillin sodium (Add before dividing the medium into plates) |
0.5 mL |
2.6.2 Experimental procedures
(1)Place the second time selecting medium in the laminar flow cabinet.
(2)Pick out those yellow callus that generate after two times of selection. Put them on the differentiation medium.
(3)Seal plates with parafilm.
(4)Culture under the condition of 14 hours light and 10 hours dark a days, for about 25 days.
(5)After 25 days, green spot will appear on the surface of the resistant callus.
(6)Place the differentiation medium in the laminar flow cabinet.
(7)Choose callus which has green spot. Transfer them to a new differentiation medium.
(8)Seal cuture bottles.
(9)Culture under the condition of 14 hours light and 10 hours dark a days, for about 30 days.
2.7 Rooting the seedling
2.7.1 Medium preparation
| Component | Dosage |
|---|---|
| 10×N6 macroelement solution | 50 mL |
| 1000×MS microelement solution | 0.5 mL |
| 100×N6vitamin solution | 5 mL |
| 100×Ferric salt solution | 5 mL |
| 0.1 mg/mL NAA | 5 mL |
| Sucrose | 10 g |
| Add distilled water until the medium volume reach 500 mL | |
| Blend with magnetic stirrer | |
| Adjust medium pH to 5.8 with 1 mol/L NaOH | |
| Phytagel | 2 g |
| 1000×Hygromycin B (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Cefazolin sodium (Add before dividing the medium into plates) |
0.5 mL |
| 1000×Carbenicillin sodium (Add before dividing the medium into plates) |
0.5 mL |
2.7.2 Experimental procedures
(1)Place the differentiation medium in the laminar flow cabinet.
(2)Carefully pick the seedling out with a sterile tweezer. Transfer it to rooting medium.
(3)Seal culture bottles.
(4)Culture under the condition of 14 hours light and 10 hours dark a days, for about 20 days.
2.8 Hardening the seedling
2.8.1 Experimental procedures
(1)Choose seedling where are 8 cm high and grown strong roots.
(2)Open the lid of culture bottle. Add appropriate amount of distilled water.
(3)Culture in the greenhouse for 7 days.
2.9 Transplanting the seedling
2.9.1 Experimental procedures
(1)After hardening the seedling, take the seeding out and remove the medium on its roots with running water.
(2)Transplant the seedling to soil. Make sure that the seedling isn’t submergence or insolated.
Extraction and detection
1. The method of extracting the astaxanthin in rice endosperm
1. Take 0.1 g rice seeds into mortar. After grinding it into powder on the ice, add 2 mL of methanol in mortar, and continue to grind for 5 minutes.
2. Transfer the powder to a 2 mL centrifuge tube, spin at 10000 rpm for 10 min in the dark and low temperature condition to extract
3. Centrifuge at 8000rpm at 4 ℃for 5 minutes and collect supernatant.
4. Extract the sediment with methanol repeatedly, until the precipitation become white.
5. Centrifuge again(8000rpm, 4 ℃, 5 min)and collect supernatant .
6. Merge supernatant, concentrate supernatant into powder in the dark and low temperature conditions
7. Add 600 µL methanol into dry astaxanthin extraction.
2. HPLC(High Performance Liquid Chromatography)determination of astaxanthin
After enrichment, put the samples into a 2 mL brown bottle through 0.22µm syringe-driven filter, then detect the samples by HPLC. The mobile phase is methanol: water = 95:5. The flow rate is 1 mL/min. Column temperature is room temperature. The detection wavelength is 480 nm.
3. Standard curve for the determination of astaxanthin
Dissolve accurately 10 mg of standard sample of astaxanthin in 1mL methylene chloride, and dilute with methanol to 100mL in a brown volumetric flask and get the 0.1 mg/mL solution. Dilute the above-mentioned solution with methanol into 2µg/mL, 4µg/mL, 6 µg/mL, 8 µg/mL, 10 µg/mL. Take 20µL sample to detect separately according to the conditions that mentioned above. With peak area as the ordinate, the concentration of the standard sample for horizontal, apply linear regression analysis, and get the regression equation.
4.Conclusion
The method of detecting the astaxanthin content in transgenic rice endosperm: grind transgenic rice seeds into powder on the ice, extract the sediment with methanol repeatedly, until the precipitation become white, concentrate the supernatant into powder, dissolved in methanol, use the high performance liquid chromatography (HPLC) to measure the content of astaxanthin.
References
【1】Y. J. Zhong, J. C. Huang, J. Liu, Y. Li, Y. Jiang, Z. F. Xu, G. Sandmann, F. Chen, Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. Journal of Experimental Botany. 62, 10, 3659–3669 (2011)
【2】J. C. Huang, Y. J. Zhong, G. Sandmann, J. Liu, F. Chen, Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants. Planta. 236, 691–699 (2012)
【3】J. C. Huang, Y. J. Zhong, J. Liu, G. Sandmann, F. Chen, Metabolic engineering of tomato for high-yield production of astaxanthin. Metabolic Engineering. 17, 59–67(2013)
【4】J. Breitenbach, C. Bai, S. M. Rivera, R. Canela, T. Capell, Paul Christou, C. f. Zhu, G. Sandmann, A novel carotenoid, 4-keto-a-carotene, as an unexpected by-product during genetic engineering of caroteno genesis in rice callus. Phytochemistry. 98, 85–91(2014)
