Simple and Rapid Regeneration of Shoot and Root from Micro-Callus Cultures in Nicotiana tabacum L.

Abstract

An in-vitro procedure is described for simple and rapid regeneration of shoots and roots from small calli of tobacco (Nicotiana tabacum L. cv. Xanthi). The calli induced from stem pith tissue were subcultured for 2 to 3 times at 2-week intervals. Then, the calli were crushed on a stainless-steel mesh with a stainless-steel spoon and passed through the mesh. Micro-calli of uniform size (0.5 or 1.0 mm in diameter) were obtained by this procedure. For shoot regeneration, micro-calli of 0.5 mm in diameter were suspended in a liquid MS medium supplemented with 0.5 mg l-1 IBA and 0.05 mg l-1 BA and rotation-cultured. After 7 days, the culture bottles were transferred to static culture under continuous light of 5400 lux. After 11 days of static culture, an adventitious shoot was formed in about 50% of the calli. The diameter of shoot-forming calli was 1 to 2 mm and the number of shoots per callus was usually one. For root regeneration, the micro-calli (1.0 mm in diameter) were rotation-cultured in the dark for 5 days in a liquid MS medium supplemented with 1.0 mg l-1 NAA and 0.1 mg l-1 kinetin, and then put separately on a 0.06-ml Gelrite drop containing the same medium composition and cultured in darkness. A regeneration frequency of about 45% was obtained by day 30 of microculture. The diameter of calli at root initiation was 4 to 5 mm and the number of roots per callus was usually one or two.