Abstract
BACKGROUND AND AIMS: Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. METHODS: A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. KEY RESULTS: It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. CONCLUSIONS: Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.
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Claims 6
Claim · model-written summary
Transformation-competent citrus cells occurred in callus arising from the cambial ring.
- Organ / stage
- newly formed callus growing from the cambial ringe0
- Outcome
- cells competent for transformatione0
CitrusOrganisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“cells competent for transformation are located in the newly formed callus growing from the cambial ring”
t0“Citrus Explants”
Claim · model-written summary
Auxin-rich medium increased cambial callus formation in inoculated and uninoculated citrus explants.
- Treatment
- medium rich in auxinse0
- Interactor
- Agrobacterium
.jpg?width=60)
Agrobacteriume0other - Outcome
- formation of cambial calluse0
- Direction
- more pronouncede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
.jpg?width=500)
Verbatim
e0“treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants”
t0“Citrus Explants”
Claim · model-written summary
Auxin-rich medium slowed citrus shoot regeneration in inoculated and uninoculated explants.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants”
t0“Citrus Explants”
Claim · model-written summary
Auxin-rich co-cultivation significantly increased the proportion of dividing citrus cells in S-phase.
- Treatment
- medium rich in auxinse0
- Outcome
- rate of actively dividing cells in S-phasee0
- Direction
- increasee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase”
t0“Citrus Explants”
Claim · model-written summary
Suitable co-cultivation conditions increased stably transformed citrus cells.
- Treatment
- proper co-cultivation medium and conditionse0
- Outcome
- number of stably transformed cellse0
- Direction
- highere0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants”
t0“Citrus Explants”
Claim · model-written summary
Suitable co-cultivation conditions increased regenerated transgenic citrus plants.
- Treatment
- proper co-cultivation medium and conditionse0
- Outcome
- final number of regenerated transgenic plantse0
- Direction
- increasee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants”
t0“Citrus Explants”
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