《植物生理学报》 2012, 48(10): 997-1004
通信作者：李润植；E-mail: rli2001@hotmail.com；Tel: 0354-6287191-306
摘 要：
以长春花幼叶为外植体建立了发根农杆菌介导的长春花高效遗传转化体系, 主要技术环节为: 用携带有基因表达载 体的发根农杆菌R1000侵染幼嫩叶片, 侵染的叶片外植体与发根农杆菌共培养2 d, 外植体移至除菌培养基除菌培养2~3周, 切取外植体上诱导长出的毛状根置于筛选培养基上培养1~2周, 最后对筛选出的阳性毛状根无性系进行扩繁。筛选出的阳 性毛状根经GUS染色和PCR分子鉴定表明, 该方法的发根诱导率和阳性转化率分别为82%±2.49%和100%。该转化方法所 获得的毛状根系数量大、质量高、遗传稳定且所需时间短, 明显优于现有的长春花遗传转化技术, 是长春花遗传转化的高 效便捷体系。关键词：长春花; 发根农杆菌; Ri质粒; 遗传转化; 毛状根
收稿：2012-08-24 修定：2012-09-13
资助：2012-09-13国家自然科学基金(31100235)、山西省青年科技研究基金(2010021030-1)和中国博士后研究经费(80839)
Corresponding author: LI Run-Zhi; E-mail: rli2001@hotmail.com; Tel: 0354-6287191-306
Abstract:
In this study, an efficient genetic transformation system of Catharanthus roseus with Agrobacterium rhizogenes was developed. The main steps of the system include infecting young leaf explants from the steriled seedlings of C. roseus with A. rhizogenes R1000 harboring an expression vector, and then co-culturing the infected leaf explants with the bacteria for two days, followed by disinfecting culture of the infected explants in the disinfected medium for two to three weeks. Putative transformed hairy roots were excised from the explants and further selected in the selection medium containing antibiotics. The positive hairy root lines were used for propagation in liquid medium. The GUS histochemical stain and PCR amplification of the genes in Ri plasmid and expression vector revealed that the induction rate of hairy root and positive transformation rate were 82%±2.49% and 100%, respectively. The hairy roots obtained by this protocol are high quality, vast growth and genetic stability, and are much better than those obtained by other methods. This transgenic protocol provides a valuable and efficient method for C. roseus transformation.Key words: Catharanthus roseus; Agrobacterium rhizogens; Ri plasmid; genetic transformation; hairy root
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