Details
| Original language | English |
|---|---|
| Article number | 1035 |
| Journal | Frontiers in Plant Science |
| Volume | 9 |
| Early online date | 24 Jul 2018 |
| Publication status | Published - Jul 2018 |
Abstract
Cyclamen persicum is an ornamental plant with economic relevance in many parts of the world. Moreover, it can be regarded as an applied model for somatic embryogenesis, since transcriptomic, proteomic, and metabolomic comparisons have revealed insights into this regeneration process on the molecular level. To enable gene function analyses, the aim of this study was to establish an efficient Agrobacterium tumefaciens-mediated genetic transformation protocol for C. persicum. For the first time, embryogenic callus cultures were used as a target material. The advantages of embryogenic callus are the defined and known genotype compared to seedlings, the high regeneration potential and the stability of the regenerated plants. A. tumefaciens strains EHA105 and LBA4404 were most efficient for transformation, resulting in transformation efficiencies of up to 43 and 20%, respectively. In regenerated plants, the presence of the transgenes was verified by PCR, Southern hybridization, and a histochemical GUS assay. The protocol was applied successfully to two C. persicum genotypes. Moreover, it served to transfer two reporter constructs, the auxin-responsive promoter DR5 driving the gus gene and the redox sensor roGFP2_Orp1, to the C. persicum genotypes, allowing the localization of high auxin concentrations and reactive oxygen species in order to study their roles in somatic embryogenesis in the future. For success in transformation, we regard the following factors as important: highly embryogenic cell lines, the use of Silwet® L-77 as a surfactant during co-culture, a genotype-specific appropriate selection schedule with hygromycin, and A. tumefaciens strains EHA105 and LBA4404.
Keywords
- Agrobacterium tumefaciens, Auxin, Dr5 promoter, Embryogenic callus, Ornamental plant, Redox sensor roGFP2_Orp1, Somatic embryogenesis
ASJC Scopus subject areas
- Agricultural and Biological Sciences(all)
- Plant Science
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In: Frontiers in Plant Science, Vol. 9, 1035, 07.2018.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Embryogenic Callus as Target for Efficient Transformation of Cyclamen persicum Enabling Gene Function Studies
AU - Ratjens, Svenja
AU - Bartsch, Melanie
AU - Winkelmann, Traud
AU - Mortensen, Samuel
AU - Kumpf, Antje
N1 - ACKNOWLEDGMENTS The kind provision of the DR5 promoter by Günther Scherer and of the plasmid pH2GW7:c-roGFP2-Orp1 by Markus Schwarzländer and Thomas Nietzel is highly acknowledged. The authors thank Bärbel Ernst, Ewa Schneider, and Friederike Schröder for their excellent technical assistance; Rena Becker, Björn Heinemann, Christine Weber, and Katharina Wellpott for their help in the lab; colleagues from the section Floriculture of our institute for their help with the Southern hybridization; and Bunlong Yim for his support in performing the statistical tests.
PY - 2018/7
Y1 - 2018/7
N2 - Cyclamen persicum is an ornamental plant with economic relevance in many parts of the world. Moreover, it can be regarded as an applied model for somatic embryogenesis, since transcriptomic, proteomic, and metabolomic comparisons have revealed insights into this regeneration process on the molecular level. To enable gene function analyses, the aim of this study was to establish an efficient Agrobacterium tumefaciens-mediated genetic transformation protocol for C. persicum. For the first time, embryogenic callus cultures were used as a target material. The advantages of embryogenic callus are the defined and known genotype compared to seedlings, the high regeneration potential and the stability of the regenerated plants. A. tumefaciens strains EHA105 and LBA4404 were most efficient for transformation, resulting in transformation efficiencies of up to 43 and 20%, respectively. In regenerated plants, the presence of the transgenes was verified by PCR, Southern hybridization, and a histochemical GUS assay. The protocol was applied successfully to two C. persicum genotypes. Moreover, it served to transfer two reporter constructs, the auxin-responsive promoter DR5 driving the gus gene and the redox sensor roGFP2_Orp1, to the C. persicum genotypes, allowing the localization of high auxin concentrations and reactive oxygen species in order to study their roles in somatic embryogenesis in the future. For success in transformation, we regard the following factors as important: highly embryogenic cell lines, the use of Silwet® L-77 as a surfactant during co-culture, a genotype-specific appropriate selection schedule with hygromycin, and A. tumefaciens strains EHA105 and LBA4404.
AB - Cyclamen persicum is an ornamental plant with economic relevance in many parts of the world. Moreover, it can be regarded as an applied model for somatic embryogenesis, since transcriptomic, proteomic, and metabolomic comparisons have revealed insights into this regeneration process on the molecular level. To enable gene function analyses, the aim of this study was to establish an efficient Agrobacterium tumefaciens-mediated genetic transformation protocol for C. persicum. For the first time, embryogenic callus cultures were used as a target material. The advantages of embryogenic callus are the defined and known genotype compared to seedlings, the high regeneration potential and the stability of the regenerated plants. A. tumefaciens strains EHA105 and LBA4404 were most efficient for transformation, resulting in transformation efficiencies of up to 43 and 20%, respectively. In regenerated plants, the presence of the transgenes was verified by PCR, Southern hybridization, and a histochemical GUS assay. The protocol was applied successfully to two C. persicum genotypes. Moreover, it served to transfer two reporter constructs, the auxin-responsive promoter DR5 driving the gus gene and the redox sensor roGFP2_Orp1, to the C. persicum genotypes, allowing the localization of high auxin concentrations and reactive oxygen species in order to study their roles in somatic embryogenesis in the future. For success in transformation, we regard the following factors as important: highly embryogenic cell lines, the use of Silwet® L-77 as a surfactant during co-culture, a genotype-specific appropriate selection schedule with hygromycin, and A. tumefaciens strains EHA105 and LBA4404.
KW - Agrobacterium tumefaciens
KW - Auxin
KW - Dr5 promoter
KW - Embryogenic callus
KW - Ornamental plant
KW - Redox sensor roGFP2_Orp1
KW - Somatic embryogenesis
UR - http://www.scopus.com/inward/record.url?scp=85050808921&partnerID=8YFLogxK
U2 - 10.3389/fpls.2018.01035
DO - 10.3389/fpls.2018.01035
M3 - Article
AN - SCOPUS:85050808921
VL - 9
JO - Frontiers in Plant Science
JF - Frontiers in Plant Science
SN - 1664-462X
M1 - 1035
ER -