Electric Field Assisting Low-Energy Ion Beam Mediated Gene Transfer

Based on the conventional indoor culture techniques of Arabidopsis thaliana, a new method which is composed of MS medium culture and water culture was developed. The results showed that, when the seeds were germinated on MS solid medium and then the seedlings were transferred to a water culture vessel containing 1/4 Hoagland solution, Arabidopsis was growing well. The method was obviously better than both soil culture only and water culture only.Arabidopsis calli were implanted with N⁺ ion beam at an energy of 20 keV and doses of 0-400×2.6×10¹³ ions/cm². Conventional method and TTC assay were respectively used to test the water loss rate and cell viability of N⁺-implanted calli. The results indicated that the water loss rate of calli increased with increasing dose, and an obvious linear relationship between them was found through regression analysis. Results of TTC-test indicated that, the cell viability was related to the dose of N⁺ implantation, and showed a characteristic curve shaped like a "saddle". A cubic equation was considered as the optimal curve equation by curve regression analysis. Besides these, 250×2.6×10¹³ ions/cm² was suggested as the optimum implantation dose to test the transformation efficiency.The distributions of DNA in horizontal and vertical electrophoresis chambers were analyzed qualitatively and quantitatively. The results proved that, when subjected to an electrical field, DNA molecules would migrate toward the positive pole. DNA degradation in double distilled water was also revealed.A novel, special, and efficient electrophoresis chamber was designed and fabricated for assisting low-energy ion beam mediated genetic transformation. DNA soaking and DNA electrophoresis were utilized as two approaches to deliver plasmid pBI121 into N⁺-implanted calli. The transient expression of GUS gene was used to analyse the transformation rate under different treatments. The results showed that DNA electrophoresis was significantly better than conventional DNA soaking. Strongly GUS expression in calli with DNA soaking was observed as multiple blue dots on the calli surfaces, while the calli with DNA electrophoresis showed apparent strongly expression in the form of multiple darker blue dots or streaks inside the calli. The highest expression rate (82.7%) was obtained when DNA electrophoresis time was 10 minutes, and the highest frequency of strongly GUS-positive calli with DNA electrophoresis (51.1%) was 5.8-fold higher than that of calli with DNA soaking (7.5%), although the DNA concentrations were the same at the beginning. Multiple comparisons indicated that 10-15 minutes was the optimal time for DNA electrophoresis to assist low-energy ion beam mediated gene transfer.