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Crambe abyssinica (crambe) is a new industrial oil crop that can grow on saline soil and tolerates salty water irrigation. Genetically engineered crambe in which the seed‐oil composition is manipulated for more erucic acid and less polyunsaturated fatty acid (PUFA) would be highly beneficial to industry. In this research, lysophosphatidic acid acyltransferase 2 RNA interference (CaLPAT2‐RNAi) was introduced into the crambe genome to manipulate its oil composition. The result showed in comparison with wild type, CaLPAT2‐RNAi could significantly reduce linoleic and linolenic acid content, simultaneously increasing erucic acid content. Systematic metabolism engineering was then carried out to further study CaLPAT2‐RNAi, combined with the overexpression of Brassica napus fatty acid elongase (BnFAE), Limnanthes douglasii LPAT (LdLPAT), and RNAi of endogenous fatty acid desaturase 2 (CaFAD2‐RNAi). Oil composition analysis on the tranformants' seeds showed that (a) with CaFAD2‐RNAi, PUFA content could be dramatically decreased, in comparison with BnFAE + LdLPAT + CaFAD2‐RNAi, and BnFAE + LdLPAT + CaFAD2‐RNAi + CaLPAT2‐RNAi seeds showed lower linolenic acid content; (b) BnFAE + LdLPAT + CaFAD2‐RNAi + CaLPAT2‐RNAi could increase the erucic acid content in crambe seed oil from less than 66.6% to 71.6%, whereas the highest erucic acid content of BnFAE + LdLPAT + CaFAD2‐RNAi was 79.2%; (c) although the four‐gene combination could not increase the erucic acid content of seed oil to a higher level than the others, it led to increased carbon resource deposited into C22:1 and C18:1 moieties and lower PUFA. Summarily, the present research indicates that suppression of LPAT2 is a new, promising strategy for seed‐oil biosynthesis pathway engineering, which would increase the value of crambe oil.

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

Background: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. Results: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L-1 were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L-1 kanamycin, 25 mg · L-1, then back to 10 mg · L-1) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L-1) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion. Conclusions: Present research revealed the po