Study On CBF3 Of Arabidopsis Thaliana Transfered Into Oncidium

The study reported the optimized Oncidium regeneration system. The result showed that: in medium consist of MS+6-BA3.0mg/L+NAA0.3mg/L+2%(W/V)Sucrose+0.6%(W/V)Agar, after twenty-five days then the Oncidium can produce Protocorm-like Bodies. The subculture period was 30 days and the propagate rate of original bulbous was 4 times. When reached a certain quantity, the protocorm was cut and inoculated on the medium (MS+6-BA1.0mg/L+NAA0.1mg/L+5%(V/V)CW+2%(W/V)Sucrose+0.6%(W/V)Agar),to promote the differentiation of adventitious bud. The clustered shoots were cultured continued on the medium (1/2MS+IBA1.0mg/L+NAA0.1mg/L+10%(V/V)CW+2%(W/V)Sucrose +0.6%(W/V)Agar). After culturing for 15 days, some of the shoots were rooting and after 25 days the length of the root are 1 cm or so. The rooting percentage was 95%.The study did the Oncidium transgenic research by particle bombardment method and agrobacterium tumefaciens transformation method.The sensitivity concentration of Hyg was tested for protocorm-like bodies. To the aim gene CBF3 for cold resistance, genetic transformation by agrobacterium tumefaciens was used to study the conversion percentage by the affecter of the pre-culture time, the infecting time and the co-cultivation time. The results showed that the sensitivity concentation of Hyg was 30mg/L for protocorm-like bodies. The genetic transformation rate was the highest when the explants with pre-culture for 1d were infecting with agrobacterium for 15 min and cocultivated with agrobacterium for 2 days. The protocorm-like bodies were cultured on media (MS+6-BA3.0mg/L+NAA0.3mg/L+Carb400mg/L)for about 15-20 days. After subcultured twice, the receptors were transferred onto media (MS+6-BA1.0mg/L +NAA0.1mg/L+5%(V/V)CW+Hyg30mg/L) for selection again.The study research the effect of the pre-culture time to transformation by particle bombardment method. And the result showed that the best conditions were 1 day pre-culture time and 6cm range. After the extension of cultivation for 10 days, the protocorm-like bodies were transferred onto media (MS+6-BA1.0mg/L+NAA0.1mg/L+5%(V/V)CW+Hyg30mg/L) for selection again.Testing the transgenic plants by PCR and Southern blotting hybridization analysis.There were 5 plants revealed positive responses mediated by agrobacferium fumefaciens.After propagating, PCR analysis primarily believed that three of them were genetic stable. Southern Blot with positive plants of PCR showed that CBF3 have been integrated into the Oncidium genome.There were only 2 positive plants acquired mediated by particle bombardment.However,PCR analysis showed that all of them was negative after propagating. This demonstrates that CBF3 have not been integrated into the Oncidium genome, and it needs to optimize the Genetic Transformation System.