Abstract
Vigorous organogenesis can be induced from hypocotyl and root explants of Arabidopsis thaliana using a two-step culture procedure consisting of preculture on callus-inducing medium (CIM) and subsequent culture on shoot-inducing medium (SIM) or root-inducing medium (RIM). With this culture system, we examined the influence of 5-bromo-2′-deoxyuridine (BrdU), a thymidine (dT) analogue, on plant organogenesis in vitro. Treatment with BrdU during SIM or RIM culture had negative effects on shoot and root redifferentiation over a broad range of concentrations. When explants were exposed to low concentrations of BrdU during preculture and then transferred onto BrdU-free SIM, shoot redifferentiation was accelerated significantly. At higher doses, BrdU treatment during the pre-culture inhibited shoot redifferentiation strongly in hypocotyl explants, but not in root explants. This suggests that a target of the BrdU action lies within the process of acquisition of cell proliferation competence specifically involved in hypocotyl dedifferentiation. These effects of BrdU were counteracted by the simultaneous addition of excess dT. BrdU-pretreated and untreated explants did not differ significantly in the phytohormone dependency of shoot redifferentiation. Our results provide a basis for future studies on plant organogenesis combining pharmacological analysis with BrdU as a probe and molecular genetics with Arabidopsis mutants.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- BrdU:
-
5-bromo-2′-deoxyuridine
- CIM:
-
callus-inducing medium
- 2,4-D:
-
2,4-dichlorophenoxyacetic acid
- dT:
-
thymidine
- GM:
-
germination medium
- IAA:
-
indole-3-acetic acid
- 2-iPA:
-
N 6-(Δ2-isopentenyl)adenine
- RIM:
-
root-inducing medium
- SIM:
-
shoot-inducing medium
References
Boyce R, Setlow R (1963) The action spectra for ultraviolet-light inactivation of systems containing 5-bromouracil-substituted deoxyribonucleic acid. I. Escherichia Coli 15 T-A-U-. Biochim Biophys Acta 68:446–454
Cary AJ, Che P, Howell SH (2002) Developmental events and shoot apical meristem gene expression patterns during shoot development in Arabidopsis thaliana. Plant J 32:867–877
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirement of suspension cultures of soybean root cells. Exp Cell Res 50:151–158
Gotô N, Hamada M (1988) Promotion of flowering by␣DNA base analogues and changes in acid phosphatase and peroxidase isozyme composition in dark-grown Arabidopsis thaliana. Plant Cell Physiol 29:683–688
Gratzner HG (1982) Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 218:474–475
Konishi M, Sugiyama M (2003) Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana. Development 130:5637–5647
Kuwagata M, Saito Y, Usumi K, Ono H, Nagao T (2001) Disruption of brain development in male rats exposed prenatally to 5-bromo-2′-deoxyuridine. Cong Anom 41:312–320
Luk DC, Bick MD (1977) Determination of 5′-bromodeoxyuridine in DNA by buoyant density. Anal Biochem 77:346–349
Morris SM (1991) The genetic toxicology of 5-bromodeoxyuridine in mammalian cells. Mutation Res 258:161–188
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Ogino H, Fujii M, Satou W, Suzuki T, Michishita E, Ayusawa D (2002) Binding of 5-bromouracil-containing S/MAR DNA to the nuclear matrix. DNA Res 9:25–29
Ohtani M, Sugiyama M (2005) Involvement of SRD2-mediated activation of snRNA transcription in the control of cell proliferation competence in Arabidopsis. Plant J 43:479–490
Ozawa S, Yasutani I, Fukuda H, Komamine A, Sugiyama M (1998) Organogenic responses in tissue culture of srd mutants of Arabidopsis thaliana. Development 125:135–142
Puck TT, Kao F-T (1967) Genetics of somatic mammalian cells, V. Treatment with 5-bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants. Proc Nat Acad Sci USA 58:1227–1234
Regan JD, Setlow RB, Ley RD (1971) Normal and defective repair of damaged DNA in human cells: A␣sensitive assay utilizing the photolysis of bromodeoxyuridine. Proc Nat Acad Sci USA 68:708–712
Shoji Y, Sugiyama M, Komamine A (1996) Suppression by 5-bromo-2′-deoxyuridine of transdifferentiation into tracheary elements of isolated mesophyll cells of Zinnia elegans. Plant Cell Physiol 37:401–403
Sugiyama M (2003) Isolation and initial characterization of temperature-sensitive mutants of Arabidopsis thaliana that are impaired in root redifferentiation. Plant Cell Physiol 44:588–596
Suzuki T, Michishita E, Ogino H, Fujii M, Ayusawa D (2002) Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells. Exp Cell Res 276:174–184
Thomas JC, Nessler C, Katterman F (1989) Interruption of somatic embryogenesis in Daucus carota L. by 5-bromodeoxyuridine. Plant Physiol 90:921–927
Toné S, Tanaka S, Kato Y (1983) The inhibitory effect of 5-bromodeoxyuridine on the programmed cell death in the chick limb. Develop Growth Differ 25:381–391
Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540
Yasutani I, Ozawa S, Nishida T, Sugiyama M, Komamine A (1994) Isolation of temperature-sensitive mutants of Arabidopsis thaliana that are defective in the redifferentiation of shoots. Plant Physiol 105:815–822
Acknowledgment
This work was supported in part by Grants-in-Aid from the Ministry of Education, Sports, Culture, Science and Technology of Japan (no.08740608).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License ( https://creativecommons.org/licenses/by-nc/2.0 ), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
About this article
Cite this article
Sugiyama, M., Imamura, K. Dose-, time-, and tissue-dependent effects of 5-bromo-2′ -deoxyuridine on the in-vitro organogenesis of Arabidopsis thaliana . Plant Cell Tiss Organ Cult 87, 17–25 (2006). https://doi.org/10.1007/s11240-006-9126-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-006-9126-y