| Wheat (Triticwn aestivum L.), a major world crops is a primary taiget for the application of genetic engineering to improve agronomic characteristic. In this paper, a regeneration system and a bombardment procedure suitable for wheat cultivars from Southwest of China were developed~ which reduced the damage to target tissues and shortened the selecting time. Through these systems~ report gene GUS and herbicide-resistance gene Bar were introduced into wheat, and transgenic wheat resistant to herbicide were obtained. 1. Development of regeneration systems. The mature embryos of fourteen wheat cultivam were cultured, and from which four were selected for their relatively high regeneration frequency. They were ChuanyuG4l3, 95319, Mianyang 19 and Chuanmai28, which were then used to develop regeneration systems using immature spikes and immature embryos as explants. When immature spikes were chosen, Mianyangl9 was proved to be better cultivar based on regeneration efficiency. Immature spikes of 0.44.0cm were introduced on MS medium containing 2,4-D 2mg/Land sucrose 3%, cal]i were regenerated on the MS medium supplemented with KTO.Smg/L and NAA 0.2mg/L, the regeneration frequency was 58.8%. When immature embryos were used as explant, Chuanyu G413 and 95319 were the main targets. Immature embryos were inoculated with the scutellum exposed on MS medium containing Z4-D 2mg/L, AI3AO.5mglL and sucrose 3%. Calli from immature embryos regenerated on the MS medium supplemented with Z1fl5 mg/L and NAAO.5 mg/L. Shoots rooted on 1/2MS medium containing IBA5rng/L and sucrose5%,regeneration frequency reached 55?5%. 2. Development of biolistics transformation systems for wheat The major parameters controlling DNA delivery to wheat by particle lxmteint were investigated, The results indicated that the best precipitants for coating particles containing SOtd 2.5moVL Ca (NO3)2 and 20pi 40% PEG4000, the suitable dosage of DNA and gold powder per bombardment was 0.25~tg125.tg. The vacuum pressure was about 27-28 inches Hg and acceptor materials were bombarded once per dish, with the Helium pressure of l3SOpsi. For immature spikes and immature embryos, it was found that transformation frequency was relatively high when immature spikes pre-cultured in vitro for 1 day was used as target tissues with the bombardment distance in 9cm, and immature embryos for 14 days with the bombardment distance in 12cm. The acceptor materials were cultured on osmotic medium (0.4molIL osmotic agents) 4 hours before bombardment and 16 hours after bombardment. 3. l ansformation and selection Selection system of high efficiency was determined. MIC(minimum inhibition concentration) of Basta to inhibit calli growth on MSJ medium was 10?0 mg/L, regeneration on MSZ medium Smg(L or 0 mg/L. MIC of Basta to inhibit shoots rooting on 1I2MSI medium was 3Omg/L Transformed under the modified bombardment protocol and the selection system, about 902 immature embryos of ChuanyuG4l3, 95319, Mianyangl9 and Bobwhite were bombarded, and 7 plants were proved transgenic. Transformation frequency was between 0.45?.2%, and transformation time was reduced to 3 months or so. 4. ConfirmatIon of transgenlc plants The transfonnants were confirmed by PCR, PCR-Southem hybridization and the resistance test of wheat leaves to herbicide Basta (0.001%) . The results of PCR and PCR-Southem hybridization showed that seven transforrnants gave positive signals, but the controls negative. The herbicide-resist... |
PDF Full Text Request