Production and Characterization of Nitrate Reductase Deficient Mutants in Petunia parviflora

Nitrate reductase deficient (NR) mutant lines were selected indirectly by their resistance to 100mM chlorate in cell cultures of P. parviflora. A total of 585 chlorate resistant lines were confirmed by a second passage on a high concentration of chlorate. Frequency of spontaneous mutation was $9.7{\times}10^{-7}$ in 3 month old suspension-cultured cells, and in non-selective media containing amino acids as sole nitrogen source. The frequency of mutation could be increased up to 11-fold by culture for 12 months. Out of 40 randomly selected calli, 22 were fully deficient in NR. The rest of the clones contained a decreased level of NR activity. Further characterization was carried out in 13 mutant lines which were fully deficient in NR and in 5 mutant lines containing residual (0-7.0%) NR activity, as compared to wild-type cells cultured on the same medium. The $NR^-$ mutants were tentatively classified as defective in the NR apoenzyme (nia-type; 11 mutant lines including the 5 with residual NR activity) or in the molybdenum cofactor (cnx-type; 7 mutant lines) by the XDH activity. The cnx-type could be further classified into two groups. In one group (5 mutant lines) of these, the NR activity could be partially restored by nonphysiologically high (1.0mM) molybdate in the culture medium. Both types of $NR^-$ mutants were unable to grow on minimal medium containing nitrate as sole nitrogen source, but grew well on amino acids. They also proved to be extremely sensitive to the standard medium ($MSP_1$) containing nitrate and ammonium. Shoot regeneration was obtained only in the $NR^-$ mutants, which contained residual NR activity, but they so far have failed to grow into plants.