Section 6
Chapter 5,075

Cultural studies on nitrate reductase deficient nicotiana tabacum cultivar gatersleben mutant protoplasts

Pental, D.; Cooper Bland, S.; Harding, K.; Cocking, E.C.; Mueller, A.J.

Zeitschrift fuer Pflanzenphysiologie 105(3): 219-228


Accession: 005074228

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Mutant plants of N. tabacum cv. Gatersleben (coded nia-130) lacking nitrate reductase apoenzyme activity and wild type (WT) plants were used for 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 N, 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 form WT protoplasts could be reduced from 14 wk to 8 wk by reducing the level of naphthaleneacetic acid after initial divisions. Approximately 1.8 .times. 107 nia-130 colonies were transferred from the amino acid-containing medium to selection medium containing nitrate as e sole source of N, but no nitrate utilizing revertants were observed. Mutant cells either did not revert or the reversion frequency was < 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.

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