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The development of a negative selection system for the isolation of plant temperature-sensitive auxin auxotrophs

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Summary

A protocol has been developed for the negative selection of plant auxotrophs using the nucleoside analogues BUdR and FUdR. The protocol was optimised using nitrogen-starved protoplast-derived cells of Nicotiana plumbaginifolia to simulate auxotrophy. The present results represent a significant improvement over previous reports in that: 1) The background of colonies escaping BUdR/FUdR kill is low and reproducible. 2) The protocol was improved to the point where background survival was 0.03% for non-starved cultures and 0.09% for auxin-starved cultures. 3) It was shown that UV irradiation decreases BUdR sensitivity of dividing cells and that this is overcome by increased exposure to BUdR. 4) Application of the method to auxin-starved haploid protoplast-derived cell suspensions resulted, for the first time, in the selection of temperature-sensitive (ts) auxin auxotrophs. 5) It could be demonstrated, for the first time, that the method in practice enriches for auxotrophs, in this case by a factor of 10 for auxin auxotrophs and at least 60 for ts auxin auxotrophs.

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Abbreviations

BUdR:

5-bromodeoxyuridine

FUdR:

5-fluoro-deoxyuridine

MNNG:

N-methyl-N′-nitro-N-nitrosoguanidine

NAA:

1-naphthaleneacetic acid

BAP:

6-benzylamino-purine

CFE:

colony forming efficiency

PE:

plating efficiency

ts:

temperature sensitive

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Authors and Affiliations

  1. Friedrich Miescher-Institut, Postfach 2543, CH-4002, Basel, Switzerland

    M. Suter, V. Schnebli & P. J. King

Authors
  1. M. Suter
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  2. V. Schnebli
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  3. P. J. King
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Communicated by I. Potrykus

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Suter, M., Schnebli, V. & King, P.J. The development of a negative selection system for the isolation of plant temperature-sensitive auxin auxotrophs. Theoret. Appl. Genetics 75, 869–874 (1988). https://doi.org/10.1007/BF00258047

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  • DOI: https://doi.org/10.1007/BF00258047

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