Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava

被引:79
作者
Bull, S. E. [1 ,2 ]
Owiti, J. A. [1 ]
Niklaus, M. [1 ]
Beeching, J. R. [2 ]
Gruissem, W. [1 ]
Vanderschuren, H. [1 ]
机构
[1] ETH, Dept Biol, Zurich, Switzerland
[2] Univ Bath, Dept Biol & Biochem, Bath BA2 7AY, Avon, England
基金
比尔及梅琳达.盖茨基金会;
关键词
MANIHOT-ESCULENTA CRANTZ; EFFICIENT PRODUCTION; SUSPENSION-CULTURES; PLANTS; RESISTANCE; PROTOCOL; VIRUS; LINE;
D O I
10.1038/nprot.2009.208
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking similar to 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.
引用
收藏
页码:1845 / 1854
页数:10
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