AbstractThe effect of phenylacetic acid (PAA) on rice (Oryza saliva L.) anther culture was investigated with six genotypes, using 2,4‐D as control. In the two‐step culture protocol, replacing 2, 4‐D with PAA in the induction medium did not influence callus induction but significantly improved the shoot differentiation from callus, particularly in the indica cultivar Teqing. The anther‐derived calli of all genotypes regenerated shoots directly on the callus induction medium containing PAA. Most of the directly‐regenerated plantlets had well‐developed root systems and were therefore readily transplanted into soil. The improved shoot differentiation potential and the frequency of direct regeneration depended on genotype, basal medium and PAA concentration. The one‐step green shoot regeneration frequencies obtained were 1.98% with the indica cultivar ‘129’, 1.5% with the indica × japonica hybrid ‘Teqing/02428’ (F1), and 1.98% with the indica × indica hybrid ‘Waiyin 2/C.B.’ (F1). The PAA‐based one‐step method was most effective on the anther culture of indica genotypes. Three DH populations have been constructed from hybrids (F1) via one‐step culture. PAA also enhanced the one‐step plantlet formation in rice somatic tissue culture.