Production of stably transformed cassava plants via particle bombardment |
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Authors: | P Zhang G Legris P Coulin J Puonti-Kaerlas |
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Institution: | (1) Institute for Plant Sciences, ETH-Zentrum/LFW E 17, CH-8092 Zürich, Switzerland e-mail: johanna.puonti-kaerlas@ipw.biol.ethz.ch Fax: +41 (1) 632 10 44, CH |
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Abstract: | A novel protocol, based on biolistics and regeneration via organogenesis, was developed for genetic transformation of cassava
(Manihot esculenta Crantz). The in vitro performance of cassava cultivars CMC40, MPer183 and MCol22 was evaluated, and the regeneration protocol was modified to improve
shoot production from explants for transformation experiments. Somatic cotyledons were used as a target tissue in the transformation
experiments using the Particle Inflow Gun and a plasmid containing the uidA gene in transient assays. The effect of different parameters for particle bombardment efficiency, including the amount of
DNA used, the flying distance of the projectiles and the pre- and post-plasmolysis time of the target tissue, was evaluated
and the conditions were partially optimised. Stably transformed cassava plants of cvs. MCol22 and TMS60444 were produced using
the partially optimised conditions and two different vector constructs carrying the hpt gene as the selectable marker. The selection protocol was optimised further, and a rooting test was developed for screening
the regenerants for antibiotic resistance to reduce the number of escapes obtained after primary selection. The production
of stably transformed cassava lines and the expression of the transgenes was verified by Southern blot analysis and RT-PCR.
Received: 10 December 1999 / Revision received: 12 January 2000 / Accepted: 7 February 2000 |
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Keywords: | Cassava (Manihot esculenta Crantz) Transformation Biolistics Organogenesis Rooting test |
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