Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

BMC Plant Biology

Research Article

Fabienne Lapeyre¹  Jean-Luc Verdeil¹  Hervé Etienne²  Eveline Dechamp²  Alessandra F Ribas²  Benoît Bertrand²  Philippe Lashermes³  Marie-Christine Combes³  Anthony Champion³ 

[1] CIRAD-BIOS, MRI, UMR-DAP, Plant cell imaging platform, Avenue Agropolis, 34398, Montpellier, Cedex 5, France;Centre de Coopération Internationale en Recherche Agronomique pour le Développement - Département des Systèmes Biologiques (CIRAD-BIOS), UMR-RPB (CIRAD, IRD, Université Montpellier II), BP 64501, 911 Avenue Agropolis, 34394, Montpellier, France;IRD - Institut de Recherche pour le Développement, UMR RPB (CIRAD, IRD, Université Montpellier II), BP 64501, 911 Avenue Agropolis, 34394, Montpellier, France;

关键词: Somatic Embryo;    Genetic Transformation;    Transformation Efficiency;    Embryogenic Callus;    Embryogenic Culture;   

DOI  :  10.1186/1471-2229-11-92

 received in 2010-10-20, accepted in 2011-05-19,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundFollowing genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica.ResultsWe identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.ConclusionMost progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.

【 授权许可】

Unknown   
© Ribas et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.