Abstract
Hybrid lethality system is one of the most serious barriers in wide hybridization. Hybrid plants between N. suaveolens and N. tabacum exhibit lethality and die at the cotyledonary stage. Intact seedling culture on solidified media containing cytokinins is a simple method for over-coming the lethality in this cross-combination. In order to improve the method, we cultured Fl seeds in liquid media containing cytokinins. Two types of cytokinins, purine-type (kinetin, 6-benzylaminopurine (BAP) and t-zeatin) and urea-type (N-phenyl-N'-(1, 2, 3-thidiazol-5-yl) urea (TDZ), N-(2-chlor0-4-pyrizyl)-N'-phenylurea (CPPU)), were used at various concentrations (0-10 mg/l ). In the liquid media containing 0.01-10 mg/l cytokinin, shoot-like structures were formed at the hypocotyl and the primary root of the dying seedlings. The shoot-like structures did not exhibit lethality and grew up to vigorous shoots without developing any lethal symptoms within a month. Vigorous multiple shoots developed from the shoot-like structures in the liquid media containing more than 0.2 mg/l cytokinins. The regenerated shoots were transferred to the medium containing 2 mg/l indole-3-acetic acid (IAA) to accelerate rooting. The hybrid seedlings displayed a vigorous growth without developing any lethal symptoms up to maturity in a greenhouse. Forty chromosomes were counted in root tip cells of the seedlings and morphological characters of the seedlings were intermediate between those of parental species. In this experiment, we developed a simplified method using liquid media with urea-type cytokinins and obtained more hybrid seedlings in a shorter period of time.
