BROMODEOXYURIDINE COMBINED WITH UV-LIGHT AND GAMMA-IRRADIATION PROMOTES THE PRODUCTION OF ASYMMETRIC SOMATIC HYBRID CALLI

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 destabiliz ing agents is described. Protoplasts of Nicotiana tabacum were fused w ith protoplasts of Nicotiana plumbaginifolia (Np) that carried the kan amycin-resistance and glucuronidase (GUS) genes on separate chromosome s. Prior to fusion, the Np donor protoplasts were pretreated with brom odeox3uridine and then were inactivated by treatment with iodoacetate +/-UV light +/-200 Gy gamma irradiation. Hybrids were selected on medi um containing kanamycin. The elimination of Np DNA was assessed by sco ring of the fraction of hybrid calli that expressed GUS and by dot-blo t analysis using a Np-specific probe. Gamma irradiation alone resulted in elimination of 50% of Np DNA. Pretreatment with bromodeoxyuridine (10 mu M) followed by 2.5 to 5 min UV light resulted in the eliminatio n of 35-45% of tile donor genome, but incorporation of bromodeoxyuridi ne (10 mu M) followed by 2.5 to 5 mill UV light and 200 Gy gamma irrad iation resulted in 85 to 90% elimination of Np DNA.