Plant Cell Reports

, Volume 24, Issue 8, pp 482–486

Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene

  • Wenwu Guo
  • Yanxin Duan
  • Oscar Olivares-Fuster
  • Zhencai Wu
  • Covadonga R. Arias
  • Jacqueline K. Burns
  • Jude W. Grosser
Genetic Transformation and Hybridization


Valencia orange [Citrus sinensis (L.) Osbeck] is the leading commercial citrus species in the world for processed juice products; however, the presence of thermostable pectin methylesterase (TSPME) reduces its juice quality. A long-term strategy of this work is to eliminate or greatly reduce TSPME activity in Valencia orange. Previous work resulted in the isolation of a putative TSPME gene, CsPME4, associated with a thermostable protein fraction of Valencia orange juice. To begin research designed to overexpress CsPME4 to verify the thermostability of the protein product and/or to downregulate the gene, a sense gene cassette containing a gene-specific sequence from a putative TSPME cDNA and the enhanced green fluorescent protein (GFP) as a selectable marker was constructed (M2.1). In the work reported here, M2.1 plasmid DNA was transformed (polyethylene glycol-mediated) into protoplasts isolated from an embryogenic suspension culture of Valencia somaclone line B6-68, in an effort to obtain transgenic Valencia lines. A vigorous transformed line was identified via GFP expression, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. One transgenic proembryo expressing GFP was recovered and multiple shoots were regenerated. The recovery of multiple transgenic plants was expedited by in vitro grafting. Polymerase chain reaction analysis revealed the presence of the PME gene in transgenic plants, and subsequent Southern blot analysis confirmed the presence of the eGFP gene. These transgenic plants show normal growth and minor morphological variation. The thermostability of PME in these plants will be assessed after flowering and fruit set. This is the first successful transfer of a target fruit-quality gene by protoplast transformation with recovery of transgenic plants in citrus. This method of transformation has the advantage over Agrobacterium-mediated transformation in that it requires no antibiotic-resistance genes.


Citrus Green fluorescent protein Juice quality improvement In vitro grafting 



Green fluorescent protein


Polymerase chain reaction


Polyethylene glycol


Pectin methylesterase


Thermostable pectin methylesterase


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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Wenwu Guo
    • 1
  • Yanxin Duan
    • 1
  • Oscar Olivares-Fuster
    • 2
  • Zhencai Wu
    • 3
  • Covadonga R. Arias
    • 3
  • Jacqueline K. Burns
    • 3
  • Jude W. Grosser
    • 3
  1. 1.National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular BreedingHuazhong Agricultural UniversityWuhanChina
  2. 2.Instituto Valenciano De Investigaciones AgrariasMoncadaSpain
  3. 3.Citrus Research and Education CenterUniversity of FloridaLake AlfredUSA

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