Abstract
The embryo-specific Dc3 gene promoter driving the VvMybA1 anthocyanin regulatory gene was used to develop a visual selection system for the genetic transformation of citrus. Agrobacterium-mediated transformation of cell suspension cultures resulted in the production of purple transgenic somatic embryos that could be easily separated from the green non-transgenic embryos. The somatic embryos produced phenotypically normal plants devoid of any visual purple coloration. These results were also confirmed using protoplast transformation. There was minimal gene expression in unstressed one-year-old transgenic lines. Cold and drought stress did not have any effect on gene expression, while exogenous ABA and NaCl application resulted in a minor change in gene expression in several transgenic lines. When gas exchange was measured in intact leaves, the transgenic lines were similar to controls under the same environment. Our results provide conclusive evidence for the utilization of a plant-derived, embryo-specific visual reporter system for the genetic transformation of citrus. Such a system could aid in the development of an all-plant, consumer-friendly GM citrus tree.
Highlights
In plants, the 5’ flanking regions of most protein-coding genes contain DNA sequences that interact with the basic transcription initiation complexes and various transcription factors [1]
Globular stage transgenic embryos could be identified from the non-transgenic escapes based on their coloration (Fig 1C) and 56 individual embryos were manually selected and individually placed in maturation medium (Table 2)
Transgenic lines remained visually free of reporter gene expression in the greenhouse
Summary
The 5’ flanking regions of most protein-coding genes contain DNA sequences that interact with the basic transcription initiation complexes and various transcription factors [1] These DNA sequences regulating the function of the downstream gene are termed promoters. Targeted gene expression in a transgenic plant by utilizing a tissue specific promoter can result in enhanced transgene protein production in the organ of interest and does not cause a metabolic drain in the overall plant [9, 10] This is because many tissue-specific promoters can provide tightly regulated gene expression in only the tissue of interest [11,12,13]
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