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.

Context. Class III acyl-CoA-binding proteins such as those from dicots (Arabidopsis and grapevine) play a role in defence against biotrophic pathogens. The overexpression of the monocot Oryza sativa (rice) OsACBP5 in Arabidopsis and rice has been demonstrated to enhance broad-spectrum disease resistance against selected phytopathogens in OsACBP5-overexpressing (OsACBP5-OE) lines. Aims. We aimed to develop transgenic rapid-cycling Brassica napus (B. napus-RC) and canola cv. Westar OsACBP5-OEs using tissue culture-based Agrobacterium-mediated transformation and to evaluate transgenic plants for resistance against Alternaria blight, blackleg and Sclerotinia rot diseases. Methods. Transgenic B. napus-RC and cv. Westar OsACBP5-OEs were generated through Agrobacterium-mediated transformation using Agrobacterium strain LBA4404 harbouring a plasmid with the rice complementary DNA encoding OsACBP5 driven by the cauliflower mosaic virus 35S promoter. Alternaria blight and blackleg pathogen assays were based on infecting young cotyledons, while detached leaf assay was used to test the tolerance of B. napus plants toward Sclerotinia sclerotiorum. Key results. Average transformation efficiencies of 24.2% and 29.1% were obtained for B. napus-RC and B. napus cv. Westar cotyledons respectively. OsACBP5-OE plants exhibited resistance 5 days after inoculation with Alternaria brassicae, 12 days after inoculation with Leptosphaeria maculans, and 24 h after inoculation with S. sclerotiorum. Conclusions. Transformation of B. napus-RC was shown herein to be an effective trait testing platform for canola. This study also provides an insight into the usefulness of OsACBP5 in enhancing resistance to necrotrophic phytopathogens. Implications. OsACBP5 can be overexpressed in other crops to generate pathogen-resistant varieties.