Engineering disease resistance and frost tolerance in canola (Brassica napus L.) using ACYL-COENZYME A-BINDING PROTEINS

Abstract

Fungal diseases and low temperature stress (freezing and frost) are major stresses faced by the canola (Brassica napus L.) industry in Australia. The potential for use of plant ACYL-COENZYME A-BINDING PROTEINS (ACBPs) in improving disease resistance and frost tolerance ability of B. napus was studied using rice (Oryza sativa) ACBP5 and Arabidopsis thaliana ACBP6 cDNA inserted into B. napus using Agrobacterium mediated transformation.

Rapid-cycling Brassica napus is a convenient model system for assessing genetic transformation of B. napus. In vitro regeneration and transformation of B. napus were optimised using rapid-cycling B. napus plants. Cotyledon explant tissues provided faster shoot induction and higher regeneration ability than hypocotyls. To further optimise regeneration using cotyledons, concentrations and combinations of routinely used plant growth regulators were tested. Cotyledon explant tissues grown in Murashige Skoog (MS) media supplemented with 1-naphthalene acetic acid (NAA) at 0.1 mg/L and 6-benzylaminopurine (BAP) at 1.0 mg/L, 0.01 mg/l gibberellic acid (GA3) and 5 mg/L silver nitrate produced a high plantlet regeneration efficiency of 70%. Four-day-old cotyledon explants co-cultivated in Agrobacterium inoculum at 0.1 optical density at 600 nm (approximately 0.1x109 cfu/ml) provided an average transformation efficiency of 16.5% when explants were sectioned without liquid medium, dipped for 30-60 sec in the Agrobacterium inoculum and had exposure to the initial antibiotic selection medium delayed for two weeks after co-cultivation.

Using these optimised tissue culture conditions, a novel candidate gene for canola pathogen resistance, ACBP5 from rice (Oryza sativa), was constitutively expressed under the CaMV35s promoter using plasmid pOS879, in canola grade B. napus cv. Westar and in rapid-cycling B. napus. Transgenic plants were confirmed by antibiotic selection, genotyping, western blot analysis and reverse transcription PCR. All positive independent lines were tested for resistance against two important pathogens of canola, namely Leptosphearia maculans (blackleg) and Sclerotinia sclerotiorum (Sclerotinia stem rot). The blackleg assay, which was based on 12-day-old cotyledons, showed that lesion development was significantly slower in OsACBP5 transgenic plants than the wild-type and vector control plants at 12 days post inoculation. The disease severity scores based on lesion size were also lower in transgenic plants than in control plants. Detached leaf assays for Sclerotinia resistance showed that there was a small but significant reduction in lesion size for transgenic plants compared to the control plants 24 h after infection. However, this effect on lesion size was not seen at 48 h, when disease development was more severe in both transgenic and wild-type plants. Overall, the transgenic plants benefited from the presence of the OsACBP5 cDNA, showing increased resistance to blackleg and an initial delaying of the effects of Sclerotinia infection.

Previously developed transgenic rapid-cycling B. napus plants expressing the Arabidopsis thaliana ACBP6 cDNA were further evaluated for their freezing and frost tolerance ability at the vegetative and seed-setting stages. The electrolyte leakage values recorded for the freezing and frost-treated plants indicated that the membrane damage of AtACBP6 transgenic plants was significantly lower than for the wild-type plants. The recovery of the freezing stressed vegetative stage plants was much quicker than that of the stressed wild-type plants and they displayed a higher harvest index than the non-transgenic plants. The yield potential as measured by the subsequent flower production was higher in transgenic plants than in the non-transgenic lines in frost-treated vegetative plants. Seed survival after freezing-with-frosting treatment showed that the wild-type seeds were more affected by the stress, showing less viability than the AtACBP6 transgenic plants. These findings indicate that the overexpression of AtACBP6 gene is potentially useful in making canola crops more tolerant of cold and frost events.

Overall, this study has provided useful evidence for the use of OsACBP5 as a novel candidate gene in decreasing the disease severity for blackleg and Sclerotinia infection in transgenic B. napus. Evaluation of the available AtACBP6 transgenic rapid-cycling plants further strengthened the evidence for the advantage of having the gene in mitigating the adverse effects of low temperature stress on B. napus plants. Transgenic plants displayed faster and better recovery and a higher yield potential than the wild-type plants. The outcomes of the project can be further extended by testing of these transgenes in commercial canola cultivars on a larger scale.