Immunological evidence for transfer of the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion

Abstract

A protoplast fusion experiment was designed in which the selectable marker, nitrate reductase (NR), also served as a biochemical marker to provide direct evidence for intergeneric specific gene transfer. NR-deficient tobacco (Nicotiana tabacum) mutant ‘Nia30’ protoplasts were the recipients for the attempted transfer of the NR structural gene from 50 krad γ-irradiated barley (Hordeum vulgare L.) protoplasts. Barley protoplasts did not form colonies and Nia30 protoplasts could not grow on nitrate medium; therefore, selection was for correction of NR deficiency allowing tobacco colonies to grow on nitrate medium. Colonies were selected from protoplast fusion treatments at an approximate frequency of 10-5. This frequency was similar to the Nia30 reversion frequency, and thus provided little evidence for transfer of the barley NR gene to tobacco. Plants regenerated from colonies had NR activity and were analyzed by western blotting using barley NR antiserum to determine the characteristics of the NR conferring growth on nitrate. Ten plants exhibited tobacco NR indicating reversion of a Nia30 mutant NR locus. Twelve of 26 regenerated tobacco plants analyzed had NR subunits with the electrophoretic mobility and antigenic properties of barley NR. These included plants regenerated from colonies selected from 1) co-culturing a mixture of Nia30 protoplasts with irradiated barley protoplasts without a fusion treatment, 2) a protoplast fusion treatment of Nia30 and barley protoplasts, and 3) a fusion treatment of Nia30 protoplasts with irradiated barley protoplasts. No barley-like NR was detected in plants regenerated from a colony that grew on nitrate following selfed fusion of Nia30 protoplasts. Because tobacco plants expressing barley-like NR were recovered from mixture controls as well as fusion treatments, explanations for these results other than protoplast fusionmediated gene transfer are discussed.