Improved method for the transformation of Arabidopsis thaliana with chimeric dihydrofolate reductase constructs which confer methotrexate resistance

Kemper, Elke; Grevelding, Christoph G.; Schell, Jeff; Masterson, Robert

doi:10.1007/bf00232162

Cited by 30 publications

(21 citation statements)

References 7 publications

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“…1); the plants were resistant to hygromycin and methotrexate, respectively. The F1 progeny ofthese crosses, presumably heterozygous for Ac and DsDHFR, were selected on medium (32) containing hygromycin (20 pug/ml) and methotrexate (0.1 ,ug/ml) to verify the presence of Ac and DsDHFR, or with kanamycin (100 pug/ml) to select for early excision events (12 (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis.

Grevelding¹,

Becker²,

Kunze³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Overexpression of the Activator (Ac) transposase gene in Arabidopsis thaliana resulted in a minimal germinal transposition frequency of 27% in which independent Dissociation (Ds) transposition events were observed. Molecular analysis of 45 F1 generation AciDs plants indicated that high rates of somatic excision had occurred, and independent germinal insertions were identified in F2 generation progeny plants. A tandem cauliflower mosaic virus (CaMV) promoter fused to two different Ac coding sequences signifcantly increased the rate of Ds transposition. The CaMV-Ac fusions activated single and multiple copies of two different Ds elements, DsDHFR and Ds35S-1, and reciprocal crosses resulted in similar transposition frequencies. The improved rate of independent germinal transposition observed makes Arabidopsis an ideal system for insertional mutagenesis.Arabidopsis thaliana is an excellent model plant for classical and molecular genetic studies. Current efforts toward characterizing the genome of Arabidopsis include restriction fragment length polymorphism analysis (1, 2), production and physical mapping of yeast artificial chromosome libraries (3-5), and Agrobacterium tumefaciens T-DNA insertional mutagenesis (6-8). An alternative means of isolating genes, which has been effective in many other model systems, is the use of transposable elements. In this paper, we report the development of a two-element Activator/Dissociation (Ac/ Ds) transposable element system suitable for gene tagging in Arabidopsis.The Ac/Ds transposable element system was originally discovered in maize by McClintock (9). It has since been shown that Ac transposes in Nicotiana tabacum (10) and numerous other dicotyledonous plant species (for review, see ref. 11). Its behavior in heterologous plant species has been most intensively studied in tobacco (10, 12-21), tomato (22-24), and A. thaliana (25-28). Many transposition-related properties associated with Ac activity in maize are also found in heterologous systems. For instance, the 4.6-kilobase (kb) autonomous element forms 8-base-pair duplications upon insertion (10), is capable of mobilizing receptor Ds elements (12,13,25,29), and preferentially transposes to nearby locations (16,28).To develop an efficient transposon tagging system for Arabidopsis and other plants, we have constructed a twoelement Ac/Ds system containing plant selectable markers designed to monitor the presence of Ac, Ds, and Ds-related excision events (25,30). A marked Ds element (DsDHFR) was constructed by inserting a plant selectable marker cassette, including a dihydrofolate reductase (DHFR) gene under the control of a cauliflower mosaic virus (CaMV) 35S promoter, into the central region of a Ds element. We have previously shown that the DsDHFR element transposes in tobacco and Arabidopsis, and the DHFR cassette does not decrease the frequency of Ds transposition (25). Furthermore, it was determined that approximately two-thirds of excised DsDHFR elements reinserted in transgenic tobacco protoplasts while the oth...

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Section: Resultsmentioning

confidence: 99%

High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis.

Grevelding¹,

Becker²,

Kunze³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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“…For seedling assays in vitro, seeds were surface-sterilized and cold treated at 48C for 3 d in the dark and then exposed to white light (;75 mE). Seedlings were grown at 228C on horizontal or vertical plates containing MS medium as modified by Kemper et al (1992), 3% sucrose, and 0.9% agarose (Merck) unless otherwise specified. For germination and red light/darkness experiments, sucrose was omitted from the medium.…”

Section: Plant Materials and Growth Conditionsmentioning

confidence: 99%

ArabidopsisCytokinin Receptor Mutants Reveal Functions in Shoot Growth, Leaf Senescence, Seed Size, Germination, Root Development, and Cytokinin Metabolism

et al. 2005

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We used loss-of-function mutants to study three Arabidopsis thaliana sensor histidine kinases, AHK2, AHK3, and CRE1/ AHK4, known to be cytokinin receptors. Mutant seeds had more rapid germination, reduced requirement for light, and decreased far-red light sensitivity, unraveling cytokinin functions in seed germination control. Triple mutant seeds were more than twice as large as wild-type seeds. Genetic analysis indicated a cytokinin-dependent endospermal and/or maternal control of embryo size. Unchanged red light sensitivity of mutant hypocotyl elongation suggests that previously reported modulation of red light signaling by A-type response regulators may not depend on cytokinin. Combined loss of AHK2 and AHK3 led to the most prominent changes during vegetative development. Leaves of ahk2 ahk3 mutants formed fewer cells, had reduced chlorophyll content, and lacked the cytokinin-dependent inhibition of dark-induced chlorophyll loss, indicating a prominent role of AHK2 and, particularly, AHK3 in the control of leaf development. ahk2 ahk3 double mutants developed a strongly enhanced root system through faster growth of the primary root and, more importantly, increased branching. This result supports a negative regulatory role for cytokinin in root growth regulation. Increased cytokinin content of receptor mutants indicates a homeostatic control of steady state cytokinin levels through signaling. Together, the analyses reveal partially redundant functions of the cytokinin receptors and prominent roles for the AHK2/ AHK3 receptor combination in quantitative control of organ growth in plants, with opposite regulatory functions in roots and shoots.

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“…For root growth analysis, seeds were surface sterilized for 10 min in 30% (v/v) commercial bleach and 0.02% (w/v) Triton X-100, rinsed three times with sterile, distilled water, and dried in a laminar flow hood. Sterile seeds were placed on ARA medium (Kemper et al, 1992) and cold treated for 3 to 4 d at 4°C in the dark. Square plates were incubated in vertical position for 4 d at 21°C under a 16-h light/8-h dark cycle light cycle.…”

Section: Plant Growth and Analysismentioning

confidence: 99%

The Arabidopsis ATHB-8 HD-Zip Protein Acts as a Differentiation-Promoting Transcription Factor of the Vascular Meristems

et al. 2001

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ATHB-8, -9, -14, -15, and IFL1/REV are members of a small homeodomain-leucine zipper family whose genes are characterized by expression in the vascular tissue. ATHB-8, a gene positively regulated by auxin (Baima et al., 1995), is considered an early marker of the procambial cells and of the cambium during vascular regeneration after wounding. Here, we demonstrate that although the formation of the vascular system is not affected inathb8 mutants, ectopic expression of ATHB-8 in Arabidopsis plants increased the production of xylem tissue. In particular, a careful anatomical analysis of the transgenic plants indicated that the overexpression of ATHB-8 promotes vascular cell differentiation. First, the procambial cells differentiated precociously into primary xylem. In addition, interfascicular cells also differentiated precociously into fibers. Finally, the transition to secondary growth, mainly producing xylem, was anticipated in transgenic inflorescence stems compared with controls. The stimulation of primary and secondary vascular cell differentiation resulted in complex modifications of the growth and development of the ATHB-8 transgenic plants. Taken together, these results are consistent with the hypothesis that ATHB-8 is a positive regulator of proliferation and differentiation, and participates in a positive feedback loop in which auxin signaling induces the expression of ATHB-8, which in turn positively modulates the activity of procambial and cambial cells to differentiate.

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Improved method for the transformation of Arabidopsis thaliana with chimeric dihydrofolate reductase constructs which confer methotrexate resistance

Cited by 30 publications

References 7 publications

High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis.

High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis.

ArabidopsisCytokinin Receptor Mutants Reveal Functions in Shoot Growth, Leaf Senescence, Seed Size, Germination, Root Development, and Cytokinin Metabolism

The Arabidopsis ATHB-8 HD-Zip Protein Acts as a Differentiation-Promoting Transcription Factor of the Vascular Meristems

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