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×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 withmagnetic 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
Extraction and detection
As our project is to use the rice as a bioreactor and produce astaxanthin,and we also hope that aSTARice can serve as a kind of food in human’s daily lives,so we are very concerned about the safety of aSTARice.
We believe that risks are mainly embodied in the following two aspects: one is that the encoding products of marker genes might be toxic and allergenic potentially for human or livestock. Antibiotic gene might be transferred into gut microbes’ genome of humans or animals , increasing the resistance of the microbes of the antibiotic, thus result in reducing the effectiveness of antibiotics in clinical treatment;the other one is that marker genes could be spread through pollens or other methods into other organisms,which is called genetic drift. Genetic drift can transfer antibiotic genes to weeds or other plants,which is threatening the ecological environment.
In order to reduce the effects of antibiotic marker gene, we use a technique called selectable marker-free technique in the project, so the final aSTARice is an marker-free transgenic plants(MFTPs).We design a Cre-loxP based side-direct recombinant system to knock out hygromycin phosphotransferase gene,minimizing the effect of the selectable maker gene.
In order to reduce the risk of genetic drift as well as the threat to the ecological environment, we add isolation area to surround the experimental fields.By isolating the transgenic rice,we reduce the possibility of genetic drift duel to the transfer of the pollen,sufficiently guaranteeing the safety of the project.Whats more,we are also analyzing coding products by HPLC and trying to make sure that there is no harmful products in our rice.
Rice was chose as the chassis though it isn’t included in the white line,but we checked-in with the official website in the beginning.Because we are the first team who choose rice as chassis,we have to asses the safety for rice.
Rice is the self-pollinated plant so that the gemotype in the individuals is pure and homogeneous.The probabilities that two flowers take place cross-pollinated are just 1%，and the distance of the spread of rice pollen are hardly 1meter.
The phenomena of pollen escape are the main ways which lead to the flow of exogenous gene of transgene plants.Recently,the reaches of transgene plants verified that the exogenous gene of transgene plants will flow to the same species or weeds and even the conventional species.As igemers,we took safety into consideration carefully so that we saw plentiful papers ,and that we found a conclusion that under the close distance less than 1% adjacent non-genetically modified plants take place gene flow.If we increase the distance to 5~10 meters,the probabilities decrease to 0.001%~0.0001%,which will not happen in theory.when planting rice,we follow seriously the safe regulations of transgene plantsSo we have faith that it is impossible for gene flow come up under this plant distance.
