Development of an Agrobacterium-mediated stable transformation method for industrial oilseed crop Crambe abyssinica ‘BelAnn’

Crambe abyssinica is a fast growing non-food high biomass crop with significant potential for biofuel production and for phytoremediation of heavy metal contaminated soils and sediments. We have developed an efficient Agrobacterium-mediated transformation system for C. abyssinica ‘BelAnn’ using β-glucuronidase (gus), bacterial arsenate reductase (ArsC), and γ-glutamylcysteine synthetase (γ-ECS) genes. Pre-cultured hypocotyl explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring binary vector pCAMBIA1300 containing gus, ArsC, and γ-ECS genes under the control of CaMV35S, leaf-specific SRS1p, and constitutive Act2p promoters, respectively. Following co-cultivation and selection, regenerated shoot buds were sub-cultured on MS medium containing GA3 for shoot elongation. Elongated shoots were transferred to root induction medium for 1 week. Semi-quantitative RT-PCR analysis confirmed the expression of mRNA transcripts for gus, ArsC and γ-ECS genes in T0 generation transgenic plants. Histochemical assays showed the gus expression in both vegetative and reproductive tissues of stably transformed T1 generation plants. Germinating seeds from T1 transgenic plants grown on MS medium containing hygromycin revealed a 3:1 Mendelian inheritance pattern for each transgene. Immunoblotting assays further confirmed the stable transformation and expression of the ArsC and γ-ECS proteins in T2 generation plants using anti-ArsC and γ-ECS antibodies. This method achieved an overall frequency of 50–70% regeneration and 6.7–8.3% transformation with three different gene constructs. The method showed higher transformation efficiency than the previously reported methods and will be useful for gene manipulation in crambe to enable biomaterial and biofuel production, as well as for potential oil content modification and heavy metal phytoremediation.

► Crambe is a fast growing non-food high biomass crop with significant industrial potential.
► A stable and high efficiency Agrobacterium-mediated transformation system for Crambe is needed.
► We report here a stable and high efficiency transformation system for the Crambe abyssinica cv BellAnn.
► Crambe transformation was optimized with three bacterial genes: gus, ArsC, and γ-ECS.
► An overall transformation frequency of 6.7–8.3% was achieved for C. abyssinica cv BellAnn.
► This method will be useful for gene manipulation in Crambe for biomaterials and phytoremediation.