In this study, we established the first efficient method to regenerate diploid apomict Boechera divaricarpa via shoot organogenesis. Hypocotyl explants were cultured on MS medium supplemented with plant growth regulators. The morphogenic potential of B. divaricarpa hypocotyl tissue was investigated to establish an efficient adventitious shoot regeneration system. We tested hypocotyls from 7-day-old in vitro seedlings. The effect of various concentrations of cytokinin and auxin on in vitro regeneration of these explants was also investigated. We found that callus induction and shoot regeneration were significantly affected by the concentrations and types of plant growth regulators. MS medium supplemented with 17.75 mu M benzylaminopurine and 0.53 mu M naphthaleneacetic acid gave the highest number of shoots per explant after 4 weeks in culture. We also determined the expression levels of three DNA methyltransferase genes under tissue culture conditions to understand their activities during callus induction and shoot regeneration. BdMET1 was expressed at low levels during both callus induction and shoot regeneration, while expression of BdDRM2 was high during callus induction and low during shoot regeneration. BdCMT3 was highly expressed in both hypocotyl explants and seedlings and may be a good candidate to induce epigenetic variations in diploid apomict B. divaricarpa shoots derived from tissue culture. Our study provides an efficient in vitro regeneration method and determines the expression levels during tissue culture of methyltransferase genes; it can be used as a new tool to understand the molecular biology of apomixis in B. divaricarpa.