Meiotic transmission of epigenetic changes in the cell-division factor requirement of plant cells

During the development of tobacco plants, cells undergo epigenetic changes that alter their requirement in culture for the cell-division factor cytokinin. Cultured leaf cells alternate between cytokinin-requiring (C^-) and cytokinin-independent (C⁺) states at extremely high rates of approximately 10^-2 per cell generation by a process called pseudodirected variation. Here we show that plants regenerated from most C⁺ clones express the Habituated leaf (Hl) trait, i.e., leaf tissues exhibit the C⁺ phenotype rather than the wild-type C^- phenotype in culture. This new trait then segregates as a monogenic dominant trait indicating that conversion of C^- cells to C⁺ cells is associated with a meiotically transmissible, genetic modification. Two independent mutants, Hl-2 and Hl-3, derived from C⁺ variants arising in culture were unstable in planta and reverted gametically at rates roughly comparable to pseudodirected variation in culture. Cells of the Hl-2 mutant, but not of a stable Hl-1 mutant, reverted phenotypically at high rates in culture. This revertant C^- phenotype persisted in some plants regenerated from cloned revertant lines, and then showed irregular segregation in two successive sexual generations. These results show for the first time that meiotically transmissible epimutations can occur reversibly and at high rates in culture.