Plant Cell Reports

, Volume 29, Issue 11, pp 1251–1260 | Cite as

An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus

Original Paper

Abstract

A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

Keywords

Agrobacterium tumefaciens Callus Cell suspension Citrus EGFP Mandarin Sweet orange Transformation 

Abbreviations

BAP

6-Benzylaminopurine

EGFP

Enhanced green fluorescent protein

hptII

Hygromycin phosphotransferase gene

MES

2-(N-morpholino)ethane sulfonic acid

MS

Murashige and Skoog medium

NAA

Naphthaleneacetic acid

nptII

Neomycin phosphotransferase gene

YEP

Yeast extract peptone

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Citrus Research and Education CenterUniversity of Florida/IFASLake AlfredUSA

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