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

Abstract

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.

Development of a CiFT Co-expression System for Functional Analysis of Genes in Citrus Flowers and Fruit

“…The long juvenile period of citrus, ranging from 6 to 20 years, has hampered the genetic improvement of reproductive tissues in transgenic plants. Although there have been some reports on gene introduction aimed at the improvement of citrus fruit quality (Guo et al, 2005;Li et al, 2002), only vegetative tissues in transgenic plants have been investigated so far. The introduction of the CiFT co-expression vector caused early flowering and fruiting in transgenic trifoliate orange.…”

Gene silencing: concepts, applications, and perspectives in woody plants

“…Research on leaf sorbitol silencing suggests that sorbitol distribution affects fruit quality such as starch accumulation and sugar-acid balance (Teo et al, 2006). In Citrus, the down regulation of putative thermostable pectin methylesterase genes is projected as a possible solution for the undesirable separation of juice into clear serum and particulate phase (Guo et al, 2005). In this case, RNAi could be used to achieve this goal.…”

Citrus biotechnology: Achievements, limitations and future directions

“…In order to enhance fruit juice quality of commercially important fruit Valencia orange, protoplasts isolated from embryogenic suspension cultures were successfully transformed by the use of PEG (Guo et al, 2005). Valencia, a highly marketable variety, is grown mainly for its juice, which is degraded due to the effect of thermostable pectin methylesterase (TSPME).…”