Summary
The degree of gamma- or X-ray-induced donor chromosome elimination in asymmetric somatic hybrids is highly variable. Here the beneficial use of bromodeoxyuridine and UV light as additional chromosome destabilizing agents is described. Protoplasts of Nicotiana tabacum were fused with protoplasts of Nicotiana plumbaginifolia (Np) that carried the kanamycin-resistance and glucuronidase (GUS) genes on separate chromosomes. Prior to fusion, the Np donor protoplasts were pretreated with bromodeoxyuridine and then were inactivated by treatment with iodoacetate ± UV light ± 200 Gy gamma irradiation. Hybrids were selected on medium containing kanamycin. The elimination of Np DNA was assessed by scoring of the fraction of hybrid calli that expressed GUS and by dot-blot analysis using a Np-specific probe. Gamma irradiation alone resulted in elimination of 50% of Np DNA. Pretreatment with bromodeoxyuridine (10 μM) followed by 2.5 to 5 min UV light resulted in the elimination of 35–45% of the donor genome, but incorporation of bromodeoxyuridine (10 μM) followed by 2.5 to 5 min UV light and 200 Gy gamma irradiation resulted in 85 to 90% elimination of Np DNA.
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Abbreviations
- BrdU:
-
bromodeoxyuridine (5-bromo-2′-deoxyuridine)
- GUS:
-
glucuronidase
- HPT:
-
hygromycin B phosphotransferase
- KmR:
-
kanamycin resistant
- Np:
-
Nicotiana plumbaginifolia
- NPT:
-
neomycin phosphotransferase II
- Nt:
-
Nicotiana tabacum
- UV:
-
ultraviolet
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Communicated by J. M. Widholm
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Trick, H.N., Bates, G.W. Bromodeoxy uridine combined with UV light and gamma irradiation promotes the production of asymmetric somatic hybrid calli. Plant Cell Reports 15, 986–990 (1996). https://doi.org/10.1007/BF00231601
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DOI: https://doi.org/10.1007/BF00231601