BACKGROUND: Rapid-cycling Brassica napus (B. napus-RC) has potential as a rapid trait testing system for canola (B. napus) because its life cycle is completed within 2 months while canola usually takes 4 months, and it is susceptible to the same range of diseases and abiotic stress as canola. However, a rapid trait testing system for canola requires the development of an efficient transformation and tissue culture system for B. napus-RC. Furthermore, effectiveness of this system needs to be demonstrated by showing that a particular trait can be rapidly introduced into B. napus-RC plants. RESULTS: An in-vitro regeneration protocol was developed for B. napus-RC using 4-day-old cotyledons as the explant. High regeneration percentages, exceeding 70%, were achieved when 1-naphthaleneacetic acid (0.10 mg/L), 6-benzylaminopurine (1.0 mg/L), gibberellic acid (0.01 mg/L) and the ethylene antagonist silver nitrate (5 mg/L) were included in the regeneration medium. An average transformation efficiency of 16.4% was obtained using Agrobacterium-mediated transformation of B. napus-RC cotyledons using Agrobacterium strain GV3101 harbouring a plasmid with an NPTII (kanamycin-selectable) marker gene and the Arabidopsis thaliana cDNA encoding ACYL-COA-BINDING PROTEIN6 (AtACBP6). Transgenic B. napus-RC overexpressing AtACBP6 displayed better tolerance to freezing/frost than the wild type, with enhanced recovery from cellular membrane damage at both vegetative and flowering stages. AtACBP6-overexpressing B. napus-RC plants also exhibited lower electrolyte leakage and improved recovery following frost treatment, resulting in higher yields than the wild type. Ovules from transgenic AtACBP6 lines were better protected from frost than those of the wild type, while the developing embryos of frost-treated AtACBP6-overexpressing plants showed less freezing injury than the wild type. CONCLUSIONS: This study demonstrates that B. napus-RC can be successfully regenerated and transformed from cotyledon explants and has the potential to be an effective trait testing platform for canola. Additionally, AtACBP6 shows potential for enhancing cold tolerance in canola however, larger scale studies will be required to further confirm this outcome.