Abstract: The cotyledon with petiole, 1/4 piece of cotyledonary laminae, 1/8 piece of cotyledonary laminae and segment of cotyledonary petiole from 7, 10 and 13 days old aseptic seedling of the yellow-seeded Brassica napus L. double haploid line 04K91 were cultured in vitro. The effect of seedling age and explant type on shoot regeneration frequency was explored. The ana1ysis of variance showed that seedling age had significant influence, explant type had greatly significant influence，and that seedling age×explant type had no remarkable effect on shoot regeneration frequency. Multiple comparisons suggested that cotyledon with petiole or 1/4 piece of cotyledonary laminae from 7 days old aseptic seedling had much higher shoot regeneration frequency, reaching to over 90% .