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Studies on Hairy Root and Cultured Cells Transformed by Ri Plasmid in Relation to Disease Resistance or Susceptibility

Ri质粒转化毛根及培养细胞与抗病或易感性关系的研究

基本信息

批准号：
63480045
负责人：
MAYAMA Shigeyuki
金额：
$ 3.2万
依托单位：
Kobe University (1990)Kagawa University (1988-1989)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

项目摘要

Utilization of hairy root induced by T-DNA insertion with Agrobacterium rhizogenes was studied to elucidate resistance or susceptibility mechanisms in plant disease and to induce resistance by gene-transfer.Hairy roots of melon were induced by A. Rhizogenes-infection. The hairy roots could grow vigorously on hormone-free medium. Hairy roots were differentiated into callus tissues when cultured in the presence of growth regulator, and shoots were frequently redifferentiated from the callus when the callus tissues were subcultured in the 50 ug/l of 2, 4-D and BAP. pBI121 was introduced into leaf cell by A. Rhizogenes, and both NPT II and GUS gene were expressed in developed hairy roots. Southern blot analysis of DNA from GUS-positive hairy root lines confirmed the integration of vector DNA to the plant chromosome. Callus tissues, leaves developed from shoots, and secondary hairy roots of regenerants were treated with kanamycin, and then subjected to histochemical detection of b-guluculonidase activity. The results clearly indicated that both the NPT vector and GUS genes transferred to the primry hairy roots were stably integrated and expressed at all stages of redifferentiation. Coat protein gene of Cucumber mosaic virus was introduced in melon cells through agroinfection, and the integration of CP gene was demonstrated by Southern blot analysis of DNA from transformed callus.Hairy roots of tomato were also induced by A. Rhizogenes. Transformed rate of the roots by T-DNA were over 50% of the induced. Screening of hairy roots resistant to Fusarium oxysporum lycopercisi was not successful.

研究了根农杆菌T-DNA插入诱导毛状根对植物病害的抗性或易感机制，并通过基因转移诱导抗性。根瘤菌侵染甜瓜产生毛状根。毛状根在无激素培养基上生长旺盛。在生长调节剂的作用下，毛状根向愈伤组织分化，在50 ug/l的2,4 - d和BAP中继代时，愈伤组织能频繁地向愈伤组织再分化。pBI121基因通过根状芽孢杆菌（A. Rhizogenes）导入叶片细胞，在发育的毛状根中同时表达NPT II和GUS基因。gus阳性毛状根系DNA的Southern blot分析证实了载体DNA与植物染色体的整合。用卡那霉素处理再生体的愈伤组织、从芽发育而来的叶片和次生毛状根，然后进行b-谷草苷酶活性的组织化学检测。结果表明，转移到毛状根上的NPT载体和GUS基因在毛状根再分化的各个阶段都稳定地整合和表达。通过农感染将黄瓜花叶病毒外壳蛋白基因导入甜瓜细胞，并通过转化愈伤组织DNA的Southern blot分析证实了CP基因的整合。根瘤菌对番茄毛状根也有诱导作用。T-DNA对根的转化率在诱导的50%以上。毛状根抗番茄枯萎病的筛选不成功。