Plant Cell Reports

, Volume 29, Issue 11, pp 1251–1260

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

Authors

  • M. Dutt
    • Citrus Research and Education CenterUniversity of Florida/IFAS
    • Citrus Research and Education CenterUniversity of Florida/IFAS
Original Paper

DOI: 10.1007/s00299-010-0910-0

Cite this article as:
Dutt, M. & Grosser, J.W. Plant Cell Rep (2010) 29: 1251. doi:10.1007/s00299-010-0910-0

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 tumefaciensCallusCell suspensionCitrusEGFPMandarinSweet orangeTransformation

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

Copyright information

© Springer-Verlag 2010