An efficient and high-throughput protocol for Agrobacterium-mediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.)

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• 作者：Yongbo Duan (12) yboduan@163.com
  Chenguang Zhai (3) Chenguang.Zhai@syngenta.com
  Hao Li (24) ahulihao@163.com
  Juan Li (24) lijuandahai@163.com
  Wenqian Mei (3) wenqian.mei@syngenta.com
  Huaping Gui (3) huaping.gui@syngenta.com
  Dahu Ni (24) dahuni1974@yahoo.com.cn
  Fengshun Song (12) sfs108@163.com
  Li Li (2) liliyjb@yahoo.com.cn
  Wanggen Zhang (35) zhangwanggen@sinochem.com
  Jianbo Yang (2) yjianbo@263.net
• 关键词：Oryza sativa L. – ; Phosphomannose isomerase (PMI) – ; Agrobacterium ; mediated transformation – ; Taqman analysis – ; High ; throughput production of transgenic rice plants
• 刊名：Plant Cell Reports
• 出版年：2012
• 出版时间：September 2012
• 年：2012
• 卷：31
• 期：9
• 页码：1611-1624
• 全文大小：861.6 KB
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• 作者单位：1. College of Life Sciences, Anhui Agricultural University, Hefei, 230036 Anhui, People鈥檚 Republic of China2. Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, 230031 Anhui, People鈥檚 Republic of China3. Syngenta Biotechnology (China), Co., Ltd, Beijing, 102206 People鈥檚 Republic of China4. Institute of Technical Biology and Agriculture Engineering, Chinese Academy of Sciences, Hefei, 230031 Anhui, People鈥檚 Republic of China5. China Seed Life Science and Technology Center, Wuhan, 430070 Hubei, People鈥檚 Republic of China
• ISSN：1432-203X

文摘

A number of Agrobacterium-mediated rice transformation systems have been developed and widely used in numerous laboratories and research institutes. However, those systems generally employ antibiotics like kanamycin and hygromycin, or herbicide as selectable agents, and are used for the small-scale experiments. To address high-throughput production of transgenic rice plants via Agrobacterium-mediated transformation, and to eliminate public concern on antibiotic markers, we developed a comprehensive efficient protocol, covering from explant preparation to the acquisition of low copy events by real-time PCR analysis before transplant to field, for high-throughput production of transgenic plants of Japonica rice varieties Wanjing97 and Nipponbare using Escherichia coli phosphomannose isomerase gene (pmi) as a selectable marker. The transformation frequencies (TF) of Wanjing97 and Nipponbare were achieved as high as 54.8 and 47.5 %, respectively, in one round of selection of 7.5 or 12.5 g/L mannose appended with 5 g/L sucrose. High-throughput transformation from inoculation to transplant of low copy events was accomplished within 55–60 days. Moreover, the Taqman assay data from a large number of transformants showed 45.2 % in Wanjing97 and 31.5 % in Nipponbare as a low copy rate, and the transformants are fertile and follow the Mendelian segregation ratio. This protocol facilitates us to perform genome-wide functional annotation of the open reading frames and utilization of the agronomically important genes in rice under a reduced public concern on selectable markers.