Plant Cell Reports

, Volume 23, Issue 6, pp 377–385

Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. × Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation

  • M. Kayim
  • T. L. Ceccardi
  • M. J. G. Berretta
  • G. A. Barthe
  • K. S. Derrick
Genetic Transformation and Hybridization

DOI: 10.1007/s00299-004-0823-x

Cite this article as:
Kayim, M., Ceccardi, T.L., Berretta, M.J.G. et al. Plant Cell Rep (2004) 23: 377. doi:10.1007/s00299-004-0823-x

Abstract

The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous β-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.

Keywords

Agrobacterium tumefaciensCitrus rootstock improvementExpansinGenetic transformation

Abbreviations

AS

Acetosyringone

CaMV 35S P

Cauliflower mosaic virus 35S promoter

CaMV 35S poly A

Cauliflower mosaic virus 35S poly A terminator

CB

Citrus blight

2,4-D

2,4-Dichlorophenoxyacetic acid

FMV

Figwort mosaic virus

GUS

β-Glucuronidase

GUS gene

uidA

IBA

Indole-3-butyric acid

MES

2-(N-Morpholino) ethane sulfonic acid

MSI

Inoculation medium

MSP-10M

Plasmolysis solution with 10% maltose

MSP-8S

Plasmolysis solution with 8% sucrose

NAA

α-Naphthaleneacetic acid

NOS

Nopaline synthase

NP

Nopaline synthase promoter

NT

Nopaline synthase terminator

NPTII

Neomycin phosphotransferase II

p12

Blight-associated protein p12

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • M. Kayim
    • 1
    • 2
  • T. L. Ceccardi
    • 3
  • M. J. G. Berretta
    • 1
  • G. A. Barthe
    • 1
  • K. S. Derrick
    • 1
  1. 1.Institute of Food and Agricultural Sciences, Citrus Research and Education CenterUniversity of FloridaLake AlfredUSA
  2. 2.Department of Plant Protection, Faculty of AgricultureUniversity of CukurovaAdanaTurkey
  3. 3.Celera Functional GenomicsSouth San FranciscoUSA