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NASC

The European Arabidopsis Stock Centre

Wave lines seed

Donated by

  • Niko Geldner Département de Biologie Moléculaire Végétale (DBMV), Faculté de Biologie et de Médecine, Université de Lausanne

Click here to view all 63 of these lines.

Description

Wave lines seed

This collection has wave lines distributed as seed stock, for wave lines distributed as DNA clones go to Wave lines DNA.

Wave lines, a set of distinct subcellular markers with spectrally distinct, fluorescent fusion constructs- provided in this collection as seed stock. The set provides an easy way to screen for distinct subcellular localization patterns and provides stable, non-toxic expression in transgenic plants. The constructs are under pUBQ10 promoter. The pNIGEL recombination vectors on which these constructs are based allow the generation of fusion constructs by CRE-lox recombination, using the pUNI ORF collection of the SSP consortium.

Nomenclature

The marker lines are available in four colors Blue (mCerulean), Bluegreen (mTFP1), Yellow (YFP) and Red (mCherry) The nomenclature of the seed lines is, for example: Wave 131C, Wave 131T, Wave 131Y or Wave 131R, the last letter indicating the different fluorophores.

Growth requirements in plants

The selection for plants are the following:

  • all the plants with YFP are Basta resistant (all the Y lines)
  • all the plants with mCherry are Hygromycin resistant (all the R lines)
  • all the plants with mCerulean and mTFP1 are Kanamycin resistant (all the C and T lines).

    Distribution

    • These seed stocks can all be ordered individually.
    • At present the complete collection is not available as seed stock.
    • The complete collection is distributed as DNA clones and vectors, as bacterial stabs N781613

    Individuals producing these lines

    • Niko Geldner, Valérie Dénervaud Tendon, York-Dieter Stierhof, Ulrike Mayer, Derek L. Hyman, Joanne Chory

    Plant transformation

    Plant transformation was performed essentially as described by Clough and Bent (1998), with the following modifications for increased throughput. pSOUP containing GV3101 Agrobacteria (see http://www.pgreen.ac.uk for information on pSOUP) were transformed with the recombined constructs, and then grown to high density in 50-ml cultures, allowing growth of up to 48 clones per shaker. Pellets were re-suspended into 200 ml of transformation medium, which provided sufficient volume for dipping. Recombined plasmids contain both ampicillin and kanamycin resistance. Kanamycin was used for selection in Agrobacteria.

    Wave lines selection and confirmation

    The initial screening for signals was performed on YFP-bearing Wave lines. Observation was carried out on non-selecting plates with segregating T2 seedlings. Between three and six independent transformants per construct were scored, and those with the best expression levels were chosen for propagation. The lines were selected for those that showed distinct signals resembling membrane compartment structures, and which could be propagated until T3, displaying good seed yield, consistent signals and no obvious developmental abnormalities. From those, red and blue spectral variants were generated. In these cases the lower number of transformants allowed us to score for up to 16 individual transformants per construct. This was especially necessary for the Cerulean-bearing Wave lines, which displayed much weaker signals on average than the mCherry and YFP-bearing lines. Homozygous lines were selected, and the presence of the correct insert was verified by PCR amplification/sequencing from plant tissues.

    Related links

    References

    • Geldner, N., et al. 2009. Rapid, combinatorial analysis of membrane compartments in intact plants with a multi-color marker set. The Plant Journal 59(1):169-78.PubMed ID: 19309456.