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Plant regeneration from callus-derived protoplasts of Pelargonium x domesticum
Authors:K B Dunbar  C T Stephens
Institution:(1) Department of Botany and Plant Pathology, Michigan State University, 48824 East Lansing, MI, USA;(2) Present address: USDA, ARS, SAA, REG. PL. INTRO. STN, 1109 Experiment Street, 30223-1797 Griffin, GA, USA
Abstract:A protocol for regenerating plants from callus-derived protoplasts of Pelargonium x domesticum (rega l geranium cv. lsquoMelissarsquo) has been developed. Protoplasts were isolated from leaf-derived callus tissue on MS medium supplemented with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6- benzylaminopurine, and 3.0% sucrose. This callus yielded 2.7×105 protoplasts/gram of tissue after a 6 hr incubation in an enzyme solution consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M sucrose. Protoplasts were plated at 1×105 protoplasts/ml in a mixture (1ratio1 v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk after plating. P-calli 1–2 mm3 developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse.Abbreviations NAA naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - CW Calcofluor White - FDA fluorescein diacetate
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