Abstract: The establishment of a good regeneration system is the foundation of agrobacterium- mediated genetic transformation. With cotyledons including part cotyledonary node fromBrassica napus as explants, a regeneration system was established. The content of this system was that cotyledons including part cotyledonary node from five days age of rapeseed seedlings were cut as explant and then were cultured in the MS media supplemented with 3 mg/L 6-BA, and five days later, shoot regenerated from the explant. The frequency of shoot regeneration was 100% . Based on the new regeneration system of cotyledon with cotyledonary node, aBnTFL1 RNAi vector constructed from pFGC5941 vector was transformed intoBrassica napus successfully by agrobacterium mediation in the research. The whole transformation period from seeding to getting rooted resistant seedlings was 60- 70 days. Compared with 100- 130 days period of traditional transformation based on regeneration culture of cotyledon without cotyledonary node, this new transformation method obviously shortened transformation period.