*4.7. Increasing Ascorbic Acid Induces Twinning*

In addition to being an antioxidant, Asc regulates the cell cycle by promoting G1 to S progression of cells, e.g., in the quiescent center of onion roots [102–106]. Repression of L-galactono-1,4-lactone dehydrogenase (GalLDH) expression in tobacco BY-2 cell lines resulted in 30% less Asc and a reduction in the rate of cell division and growth [107]. The ability of Asc to promote cell division had dramatic consequences when its level was elevated during early embryo development. Embryo development initiates with a transverse zygotic division to produce an apical, proembryo cell and a basal cell that gives rise to the suspensor and in most species, a single embryo develops in each seed. Increasing Asc content in tobacco by increasing DHAR expression, however, resulted in monozygotic twinning and polycotyly [108]. The twin zygotes resulted from a longitudinal instead of transverse cell division and these twin zygotes developed into embryos of equal size. Direct injection of Asc into tobacco ovaries was sufficient to induce twinning but only if delivered within the first two days after pollination during which the zygote undergoes its first division. The twinning can be understood as an Asc-induced alteration in the normal transverse division of the zygote that results in a loss of the positional cues needed for the normal differentiation of the apical cell into the embryo and the basal cell into the suspensor.

Polycotyly (*i.e.*, the development of more than two cotelydons) was also induced by Asc, either following an increase in DHAR expression or when Asc was injected at the globular stage of embryo development prior to the initiation of cotyledon development [108]. As in zygotic division, an Asc-induced alteration in cell division during the specification of cotyledon-forming fields likely is responsible for the observed polycotyly. Although Asc likely affects cell division in other tissues [102–106], the lack of a readily observable phenotype may make the effect of increased Asc content in other aspects of plant development less apparent.
