Weeks J. T., Anderson O. D., Blechl A. E.
Agricultural Research Service, United States Department of Agriculture, Western Regional Research Center, Albany, California 94710.
Plant Physiol. 1993 Aug;102(4):1077-1084. doi: 10.1104/pp.102.4.1077.
Improvement of wheat (Triticum aestivum) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation methodology. In this report, we detail a protocol for transformation of a highly embryogenic wheat cultivar, Bobwhite. Calli derived from immature embryos, 0.5 to 1 mm long, were bombarded with microprojectiles coated with DNA containing as marker genes the bar gene, encoding phosphinothricin-resistance, and the gene encoding [beta]-glucuronidase (GUS), each under control of a maize ubiquitin promoter. The bombardment was performed 5 d after embryo excision, just after initiation of callus proliferation. The ability of plantlets to root in the presence of 1 or 3 mg/L of bialaphos was the most reliable selection criteria used to identify transformed plants. Stable transformation was confirmed by marker gene expression assays and the presence of the bar sequences in high molecular weight chromosomal DNA of the resultant plants. Nine independent lines of fertile transgenic wheat plants have been obtained thus far, at a frequency of 1 to 2 per 1000 embryos bombarded. On average, 168 d elapsed between embryo excision for bombardment and anthesis of the T0 plants. The transmission of both the resistance phenotype and bar DNA to the T1 generation verified that germline transformation had occurred.
目前,生物技术方法对小麦(普通小麦)的改良受到缺乏高效可靠转化方法的限制。在本报告中,我们详细介绍了一种用于转化高胚性小麦品种“博比特”的方案。从长度为0.5至1毫米的未成熟胚诱导产生的愈伤组织,用包被有DNA的微粒进行轰击,该DNA含有作为标记基因的bar基因(编码膦丝菌素抗性)和编码β-葡萄糖醛酸酶(GUS)的基因,每个基因都受玉米泛素启动子的控制。轰击在胚切除后5天进行,恰好在愈伤组织增殖开始之后。在含有1或3毫克/升双丙氨膦的情况下,植株生根的能力是用于鉴定转化植株的最可靠选择标准。通过标记基因表达分析以及在所得植株的高分子量染色体DNA中存在bar序列,证实了稳定转化。到目前为止,已获得了9个独立的可育转基因小麦株系,轰击的1000个胚中转化频率为每1000个胚产生1至2个株系。从轰击的胚切除到T0植株开花平均经过168天。抗性表型和bar DNA向T1代的传递证实发生了种系转化。