根癌农杆菌介导的橡胶树花药愈伤组织遗传转化体系的建立

为建立根癌农杆菌介导的橡胶树遗传转化体系,以花药愈伤组织为受体,研究了农杆菌菌株、共培养温度、共培养时间、乙酰丁香酮(AS)等因素对遗传转化效率的影响。结果表明,农杆菌菌株、共培养温度和共培养时间对转化效率具有显著影响,AS不影响转化效率。2.2万个花药愈伤组织经EHA105菌株侵染后,转入未添加AS的培养基,22℃共培养6d,通过50mgL-1卡那霉素抗性筛选、叶片GUS染色、uidA和NptII基因PCR特异扩增、PCR产物测序及NptII基因Southern检测,鉴定出11株转基因植株,并通过嫁接和次生体胚发生,获得来自8个转基因株系的681株转基因植株,移栽成活253株。

Establishment of Agrobacterium tumefaciens-mediated Anther Calli Transformation System in Hevea brasiliensis

Developping a new Hevea variety by crossing breeding will take 20-30 years, but genetic transformation provides an effective alternative for shortening breeding cycle. Until now, two kinds of recipient materials, anther compact embryogenic calli (ACEC) and maintained friable embryogenic calli (MFEC), have been used successfully for Hevea genetic transformation. However, few transformed plants are produced from ACEC-derived embyos, and plants regenerated from MFEC are abnormal, showing that they have poorer performance in growth, yield and disease resistance than the budded control. In order to establish Agrobacterium tumefaciens-mediated Hevea factors influencing transformation frequency, including strains of Agrobacteriumembryos regeneration. GUS transient expression frequency was significantly affected by Agrobacterium strains and co-culture temperature/time, which was 5.07 blue spots/callus for EHA105, higher than that for GV3101 and LBA4404. The highest GUS transient expression frequency was 8.43 and 19.10 blue spots/callus under 22℃ co-cultured temperature and 6 days co-cultured time respectively. Twenty-two thousand ACEC were transformed by the strain EHA105 with pCAMBIA2301, and then transferred to the medium without AS and incubated at 22℃ for 6 days in the dark. Subsequently, 11 transformants were identified by GUS staining, PCR amplification of target genes, sequencing of PCR products and Southern hybridization. Among them, three planted directly in the field died; three as scions were budded onto seedlings, producing five budded transgenic plants; five were multiplicated at embryo phase by secondary embryogenesis, producing 676 transgenic plantlets, among which 248 survived in the field. The results showed that the present transformation system and the plant propagation by transgenic embryos secondary embryogenesis are efficient for the production of transgenic rubber plants., co-culture temperature/time and acetosyringone (AS), and the methods for anther calli transformation system, we surveyed the