Cultural Studies on Nitrate Reductase Deficient Nicotiana tabacum Mutant Protoplasts

Abstract

Summary Mutant plants of Nicotiana tabacum cv Gatersleben (coded nia -130) lacking nitrate reductase apoenzyme activity and wild type (WT) plants were used for the isolation of leaf mesophyll protoplasts. Mutant plants grafted onto a WT stock gave yields of protoplasts comparable to those from WT plants. Protoplasts and cell colonies from nia -130 plants did not grow in media containing nitrate salts as the sole source of nitrogen, while WT protoplasts divided and proliferated on such a medium. Mutant protoplasts underwent division on a medium supplemented with amino acids and produced cell colonies which regenerated plants (12-14 wk). The total time for plant regeneration from WT protoplasts could be reduced from 14 wk to 8 wk by reducing the level of naphthaleneacetic acid (NAA), after initial divisions. Approximately 1 · 8 x 10 7 nia -130 colonies were transferred from the amino acid containing medium to selection medium containing nitrate as the sole source of nitrogen, but no nitrate utilizing revenants were observed. Mutant cells either did not revert or the reversion frequency was less than 10 -7 . One or more WT colonies were mixed with mutant cultures and plated in the selection medium. The recovery of WT colonies was best on nitrate medium with low levels of auxin. The possibility of recovering rare nitrate utilizing events, and good plant regeneration capability, may make this protoplast system ideal for cell fusion and genetic transformation studies.