Engineering oilseed crops for industrial purposes requires a suitable crop that does not outcross to any food oilseed crop, thus eliminating problems of gene flow. Crambe abyssinica is such a dedicated crop as it does not hybridize with any existing food oilseed crops. However, lack of regeneration and transformation protocols has limited the use of C. abyssinica in genetic manipulation studies. In this study, efficient regeneration and transformation protocols for Crambe have been developed. Hypocotyls of C. abyssinica cv. Galactica were incubated on a Murashige and Skoog medium supplemented with various plant growth regulators (PGRs). Among the different PGR combinations tested, 10 lM thidiazuron and 2.7 lM a-naphthaleneacetic acid promoted highest frequency of regeneration, up to 60%. Among six Agrobacterium stains evaluated, each harbouring the cloning vector containing the neomycin phosphotransferase (nptII) and b-glucuronidase (gus) genes. EHA101 and AGL-1 yielded the highest transformation frequencies of 1.3 and 2.1%, respectively. Putative transgenic lines were recovered, and confirmed as transgenic by Southern blot analysis. Subsequently, Agrobacterium-mediated transformation of hypocotyls of cv. Galactica with constructs harbouring the wax synthase and fatty acid reductase genes have also successfully recovered confirmed transgenic plants carrying these transgenes.

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We have developed an efficient rooting protocol using half-strength Lepoivre medium for studying AR formation in stems, traced back the cellular AR origins in stems, and correlated expression of rooting-related genes with root initiation sites.

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The wild species field cress (Lepidium campestre), belonging to the Brassicaceae family, has potential to be developed into a novel oilseed- and catch crop, however, the species needs to be further improved regarding some important agronomic traits. One of them is its low oil content which needs to be increased. As far as we know there is no study aiming at increasing the oil content that has been reported in this species. In order to investigate the possibility to increase the seed oil content in field cress, we have tried to introduce the Arabidopsis WRINKLED1 (AtWRI1) or hemoglobin (Hb) genes from either Arabidopsis thaliana (AtHb2) or Beta vulgaris (BvHb2) into field cress with the seed specific expression. The hypothesis was that the oil content would be increased by overexpressing these target genes. The results showed that the oil content was indeed increased by up to 29.9, 20.2, and 25.9% in the transgenic lines expressing AtWRI1, AtHb2, and BvHb2, respectively. The seed oil composition of the transgenic lines did not significantly deviate from the seed oil composition of the wild type plants. Our results indicate that genetic modification can be used in this wild species for its fast domestication into a future economically viable oilseed and catch crop.

BackgroundLepidium campestre is an undomesticated oilseed species with a great potential to become a new crop for both food and industrial feedstocks production. Genetic modification is needed for further improving the oil quantity and quality of Lepidium. Studies on in vitro shoot regeneration of Lepidium are very limited and there is no transformation protocol available.ResultsWe have investigated the effects of different factors, especially the type, concentration and combination of plant growth regulators (PGRs) on in vitro shoot regeneration of Lepidium. The results showed that the 2,4-D treatment was crucial to shoot regeneration from different explants. The duration of 2,4-D exposure between 2-4 days did not show significant difference in shoot regeneration, while the effect of 2,4-D concentration varied greatly depending on the type of explants and cytokinins used, for example, the low concentration of 2,4-D combined with TDZ significantly increased the regeneration frequency of hypocotyls. Cotyledon and hypocotyl explants responded differently to cytokinin, for example, TDZ was more effective than zeatin in promoting shoot regeneration from hypocotyls, but did not affect the regeneration of cotyledons which was more affected by high concentration of zeatin. The results also showed that NAA was not effective for shoot regeneration. Germination in light increased the regeneration frequency compared to that in dark. After optimization of the different conditions, an efficient regeneration protocol was developed with the regeneration efficiency of 92.7%. Using this protocol, the transformation frequency of 6% in average was achieved. The presence of transgenes in the transgenic lines was confirmed by GUS staining, PCR and Southern blot analyses.ConclusionThrough systematic investigation of important factors affecting in vitro shoot regeneration, we have developed an efficient regeneration and transformation protocol for the genetic modification of Lepidium campestre. The method may also be applied to the related species.