Timing of the G1/S transition in tobacco pollen vegetative cells as a primary step towards androgenesis in vitro.

Mid-bicellular pollen vegetative cells in tobacco escape from G1 arrest and proceed to the G1/S transition towards androgenesis within 1 day under glutamine starvation conditions in vitro. In the Nicotiana tabacum pollen culture system, immature pollen grains at the mid-bicellular stage can mature in the presence of glutamine; however, if glutamine is absent, they deviate from their native cell fate in a few days. The glutamine-starved pollen grains cannot undergo maturation, even when supplied with glutamine later. Instead, they undergo cell division towards androgenesis slowly within 10 days in a medium containing appropriate nutrients. During the culture period, they ought to escape from G1 arrest to proceed into S phase as the primary step towards androgenesis. However, this event has not been experimentally confirmed. Here, we demonstrated that the pollen vegetative cells proceeded to the G1/S transition within approximately 15-36 h after the start of culture. These results were obtained by analyzing transgenic pollen possessing a fusion gene encoding nuclear-localizing GFP under the control of an E2F motif-containing promoter isolated from a gene encoding one of DNA replication licensing factors. Observations using a 5-ethynyl-2'-deoxyuridine DNA labeling and detection technique uncovered that the G1/S transition was soon followed by S phase. These hallmarks of vegetative cells undergoing dedifferentiation give us new insights into upstream events causing the G1/S transition and also provide a novel strategy to increase the frequency of the androgenic response in tobacco and other species, including recalcitrants.