Culture surface and exogenous putrescine-altered shoot growth pattern in mannitol- and cadmium chloride-pretreated callus of Cymbidium Via del Playa “Yvonne”

Abstract

Evidence for the link between the surface of culture medium and abnormal shoot formation in the presence of exogenous putrescine is presented. Little is known about the role of putrescine in rapid shoot regeneration in the callus of orchids pretreated with mannitol and cadmium chloride (CdCl2). Ultrastructural analysis of putrescine-altered CdCl2- and mannitol-treated callus after 4 wk of culture showed the presence of numerous oil droplets, rough endoplasmic reticulum, and dictyosomes. Callus pretreated with mannitol and CdCl2 but without exogenous putrescine showed the presence of amyloplasts, condensed mitochondria, and active vacuole within 12 h of pretreatment. This study not only establishes a new method to achieve rapid regeneration of Cymbidium callus but also describes different types of shoots on the basis of its contact with the surface of the culture medium. Histologically, two types of cells in the apical meristem are defined. One type of cells has compromised volume which was lost upon division and is represented by small deeply stained cells of the shoots regenerated from the upper portion of the callus and is considered normal. The other type is represented by highly vacuolated and less deeply stained cells of the shoots regenerated from the lower portion of the callus in contact with the medium and is considered abnormal in this study. To avoid such abnormalities, frequent subculture of callus halves which were obtained by a median transverse cut before the regenerants (PLBs) were converted to shoots is recommended. This new methodology was successful in achieving rapid regeneration of uniform shoots from callus.