[Quick testing-technology of transgenic rice with npt- screen marker gene].
By using npt-gene as assistant selection-marker, treating Japonica rice Zhonghua 9 [ZH9 (CK)] and transferred lysozyme gene rice (the donor rice is Japonica rice Zhonghua 9) [ZH9(R)] with antibiotics, we built a system of quickly testing transgenic rice offspring. Detached leaves of ZH9(R) and ZH9(CK) were treated by Kanamycin (0, 400, 450, 500, 550, 600 and 700 mg/L) and G418 (0, 40, 60, 80, 100, 150 and 200 mg/L) with different concentrations. The result show effect of Kanamycin is not evident and G418 is the best antibiotics to test transgenic rice with npt- gene. The data showed that 80 mg/L G418 (treating 4 d) was optimal to test transgenic rice. Further study was done with seeds, young embryos and seedlings by G418 testing: the alive seeds were cultured in the culture dishes which filled with a series of G418 solution with different concentration of 0, 100, 150, 200, 250, 300 and 350 mg/L; in the tissue culture room, the germinating embryos were inoculated in the culture medium (1/2 MS+0.5 mg/L 6-BA+1.5% sucrose) which contains G418 with 0, 150, 200, 250 and 300 mg/L concentration; the aseptic seedlings were inoculated in the the same culture medium (1/2 MS+0.5 mg/L 6-BA+1.5% sucrose) which contains G418 with 0, 100, 150, 200 and 250 mg/L concentration. The conclusions indicated that 300 mg/L (treating 7 d) was the critical concentration to test seeds of transgenic rice; 200 mg/L (treating 10 d) was the critical concentration to test young embryo of transgenic rice; 150 mg/L (treating 12 d) was the critical concentration to test seedlings of transgenic rice. Two primers were designed based on npt- and lysozyme gene sequences. PCR technology confirmed the above detection system. The preliminary results showed npt-was tightly linked with lysozyme gene. Above confirmed critical concentrations were applied to test detached leaves, seeds, young embryos and seedlings of transferred generations. The effect was very obvious. It is convenient, intuitionistic, and exact way that aparting the positive plants from the mixture of transgenic positive and negative plants with G418.
Wang
Zi-Xuan
ZX
Key Laboratory of Cell Engineering, Hunan Agricultural University, Changsha 410128, China. zixuanwang20@126.com
Yi
Zi-Li
ZL
Wang
Zhi-Cheng
ZC
Jiang
Jian-Xiong
JX
Qin
Jing-Ping
JP
Zhang
Hao
H
Tan
Yan-Ning
YN
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D015415:Biomarkers; D019411:Clinical Laboratory Techniques; D018506:Gene Expression Regulation, Plant / Q000187:drug effects*; D005839:Gentamicins / Q000494:pharmacology*; D007612:Kanamycin / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000235:genetics; D012275:Oryza; D018515:Plant Leaves / Q000187:drug effects*; D030821:Plants, Genetically Modified / Q000235:genetics*; D036226:Seedlings / Q000187:drug effects / Q000254:growth & development; D012639:Seeds / Q000187:drug effects*
D001426:Bacterial Proteins; D015415:Biomarkers; D005839:Gentamicins; D026901:Membrane Transport Proteins; C117830:npt2 protein, Chlamydia trachomatis; D007612:Kanamycin; C010680:antibiotic G 418