Genetic engineering in the oilseed crop camelina: establishment and application

In vitro regeneration and Agrobacterium-mediated genetic transformation are essential methods in plant and breeding research. However, the opportunities for applying modern biotechnological methods to some crops are limited by protocols that are still too inefficient. This particularly affects the utilization of the considerable potential of genome editing. To overcome these limitations in the increasingly important oilseed camelina (Camelina sativa), immature zygotic embryos were used for in vitro regeneration, as it was assumed that tissue from this explant type has a higher totipotency compared to conventionally used, more differentiated plant parts. After the formation of adventitious shoots and their further development into plants using the experimental accession Cam139 had indeed been achieved with high efficiency, the underlying initial developmental processes were investigated at the cellular level using microscopic preparations, and it was determined that the regenerative structures originate in the outermost layers of the embryonic hypocotyl. In addition, this regeneration principle, which is largely unexplored for dicotyledonous plants, has also been successfully applied to the current camelina variety Ligena. The newly developed regeneration method was then used to establish Agrobacteriummediated transformation for camelina. Several influencing factors were varied in this experimental work, including the targeted mechanical wounding, the duration of the pre- and co-culture and the concentration of the selection agent hygromycin. This method also proved to be applicable to current varieties, with transformation efficiencies of up to 17% (based on the number of cultivated explants) being achieved. Finally, the newly established transformation method was used to knock out the gene encoding the fatty acid-modifying enzyme FATTY ACID ELONGASE 1 (FAE1) by targeted mutagenesis using CRISPR-associated (Cas) 9 endonuclease. To this end, a protoplast-based method of functional validation of in silico pre-selected Cas9 target motifs and their cognate guide (g)RNAs was developed. Using this test, mutations were detected in all three preselected FAE1-specific target motifs of all three homeologous camelina subgenomes. In the deep-sequencing of target region-specific PCR amplicons of genomic protoplast DNA, up to 15% of the sequencing reads proved mutated. After using the same construct to produce stably transgenic plants, mutation events were also detected in all three FAE1-specific target motifs of all three homeoalleles. Thanks to this high efficiency, 8 out of 9 primary transgenic plants carried mutations in all three FAE1 homeoalleles. Consequently, triple-homozygous fae1 knockout mutants could already be identified in the first selfing generation. The fatty acid profiles of these knockout lines showed that the nutritionally particularly valuable proportions of linoleic acid (C18:1) and a-linolenic acid (C18:2) were significantly increased and that erucic acid (C22:1), which leads to impaired oil quality, was reduced to barely detectable traces. Overall, this work comprises significant advances from a basic biotechnological exploration of the oilseed camelina to the successful use of genome editing, resulting in the development of elite lines with unprecedented high oil quality as an important prerequisite for a healthy diet.