Changes in symplastic permeability during adventitious shoot regeneration in tobacco thin cell layers

Thin cell layer (TCL) explants of tobacco (Nicotiana tabacum L.) were cultured in either a regeneration medium that resulted in formation of adventitious vegetative shoots or a non-regeneration (control) medium that maintained the TCLs but did not promote shoot formation. Microinjections were conducted on epidermal cells at 1- or 2-day intervals during the culture period (14 days) and also on meristematic regions as they appeared in regenerating TCLs. A fluorescein isothiocyanate-labelled peptide (F(Glu)₃ MW 799) was used to assess the permeability of the symplast during adventitious shoot regeneration. A period of increased symplastic movement of F(Glu)₃ was detected during day 2 of culture and was significantly greater in regenerating TCLs than in non-regenerating TCLs. This corresponded to the period of the first cell divisions and represents the re-initiation of a meristematic type of symplastic linkage between epidermal cells. A smaller increase in cell-to-cell movement within non-regenerating TCLs indicated a possible stress response as a factor in these changes. Movement of F(Glu)₃ throughout the epidermal symplast of regenerating TCLs returned to pre-culture levels by the time of shoot primordia formation. F(Glu)₃ movement was further down-regulated in non-regenerating TCLs, with a high degree of cell isolation observed. Within newly formed shoots, symplastic movement of F(Glu)₃ cycled between high and low levels.