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Arabidopsis shoots regenerate from root explants through a two-step process consisting of pre-incubation on an auxin-rich callus induction medium (CIM), followed by transfer to a cytokinin-rich shoot induction medium (SIM). The auxin receptor gene TIR1 was up-regulated when explants were transferred to SIM. The CIM pre-incubation is required for its up-regulation. The tir1-1, TIR1 knockdown mutant, reduced the efficiency of shoot regeneration in tissue culture, while its over-expression mutant significantly improved efficiency. TIR1 promoter::GUS fusion analysis demonstrated that TIR1 expression was in the shoot and the newly emerging leaves. After 10 d on SIM, several cytokinin related genes (CDKB1;1, CKS1, IPT4 and ARR15), which associate with shoot regeneration, were up-regulated in plants over-expressing TIR1 and some of these were down-regulated in the tir1-1 mutant. Thus, TIR1 appears to be involved in regulating shoot regeneration.