Regeneration of Plants from Callus-derived Protoplasts of Salpiglossis1

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Abstract
Callus cells were used as the cell source for isolating protoplasts of Salpiglossis sinuata L. Callus was initiated on the cut edges of leaf sections cultured on Uchimiya and Murashige (UM) medium under 24 μEm-2s-1 at 30°C. Friable callus from subcultures on UM was subjected to an enzymatic solution of 1% Driselase, 0.75% Pectinase, 2% Cellulase R-10, and 8% mannitol in CPW salts, and incubated at 50 rpm, for 4-5 hr to release protoplasts. Following washing, counting, and dilution, protoplasts were plated in liquid Murashige and Skoog medium (MS) modified by deleting the ammonium ions and adding (mg/liter): 250 L-glutamine, 0.1 L-serine, 2.0 thiamine, 0.5 indoleacetic acid (1AA), 1.0 2,4-D, 0.5 6-benzylamino purine (BA) and 9% mannitol. Within 2-5 days, cell wall synthesis and the first cell division occurred. Green-yellowish colonies, 2 to 3 mm in diameter, formed in 2.5 months and were transferred to MS + 1.0e mg/liter N6-isopentenyladenine (2iP), where shoot primordia were evident within 21 days. After full development and elongation of shoots, they were dipped in 1000 ppm indolebutyric acid (1BA) and placed in MS + 0.001 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D) to initiate roots. Rooting was carried out at 22±2°C under 80µm-2s-1 Cool White fluorescent tubes. Nine of 10 regenerated plants examined cytologically were 2n = 44, normal for the species, and 1 plant was 2n =88.

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