Abstract: To obtain various genetic resources，interspecific tri-parental somatic hybridization among Brassica campestris L. ssp. pekinensis（2n = 20，AA），B. oleracea L. var. varitalica（2n = 18，CC），and B. juncea L. Czern（2n = 36，AABB）was performed. Protoplasts were isolated from hypocotyls and cotyledons，and fused using the 40% polyethylene glycol（PEG）. Fused protoplast were cultured in modified K8p medium supplemented with 0.2 mg · L-1 2，4-D，0.5mg · L-1 6-BA，0.1 mg · L-1 NAA，and 0.1 mg · L-1 kinetin，and 0.4 mol · L-1 mannitol as osmoticum. At the 8–10-cell stage，divided cells were transferred to Kao’s basal medium supplemented with 0.3 mol · L-1 sucrose as carbon source，and 0.15% agarose，2 mg · L-1 6-BA，2 mg · L-1zeatin，1 mg · L-1 NAA，0.5 mg · L-1 kinetin for callus proliferation. After 35 days，when small calli reached 2–3 mm in diameter，calli were transferred to regeneration medium（MS + 5 mg · L-1 zeatin + 2 mg · L-1 IAA；MS + 2 mg · L-1 zeatin + 2 mg · L-1 IAA；MS + 0.5 mg · L-1 6-BA + 0.5 mg · L-1NAA）. A total 96 regenerated plants were obtained from 932 calli. Several phenotypes were obtained in the somatic hybrids，showing differences in leaf morphology，flower size and color. Most of the plants showed intermediate characteristics between two or more of the parental species. Cytoplasmic male sterility（CMS）identified by polymerase chain reaction（PCR）amplification using Ogura CMS specific primers. Putative somatic hybrids were identified by cytological and morphological analyses. The chromosome numbers of the somatic hybrids varied from 38 to 64，which was less than the sum of the parental lines. The somatic hybiridity was confirmed by Genomic in situ hybridization（GISH）and amplified fragment length polymorphism（AFLP）analysis.

Key words: Brassica, interspecific, tri-parental somatic hybridization, protoplast, plant regeneration, morphology, genomic in situ hybridization