Genetically engineering Crambe abyssinica- A potentially high-value oil crop for salt land improvement

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

Formation Control Algorithm of Agents Based on Earth Mover’s Distance

Massive sport, such as unmanned aerial vehicle performance, often needs fast and efficient calculation of formation morphing and individual path planning. This paper introduces a novel fast formation control method of a crowd. First, we get the agents’ location in a 2D polygon with centroidal Voronoi tessellation and L-BFGS techniques. Then, we transform crowd formation shapes with a global shortest motion path pair assignment using earth mover’s distance algorithm. Finally, the repulsing force between agents and obstacles is calculated based on the recursive velocity observer method control agents’ motion. Extensive experimental results show the effectiveness and usefulness of our algorithm in 2D group formation transformation

Correlation, Regression and Path Analyses of Seed Yield Components in Crambe abyssinica, a Promising Industrial Oil Crop

In the present study correlation, regression and path analyses were carried out to decide correlations among the agro- nomic traits and their contributions to seed yield per plant in Crambe abyssinica. Partial correlation analysis indicated that plant height (X1) was significantly correlated with branching height and the number of first branches (P <0.01); Branching height (X2) was significantly correlated with pod number of primary inflorescence (P <0.01) and number of secondary branches (P <0.05) and negatively correlated with number of first branches (P <0.01); Number of first branches (X3) was significantly correlated with number of secondary branches (P <0.01), pod number per plant and 1000-grain weight (P <0.05); Number of secondary branches (X4) was significantly correlated with seed yield per plant (P <0.05); Pod number per plant (X7) was significantly correlated with seed yield per plant (P <0.01) and negatively correlated with 1000-grain weight (P <0.01); 1000-grain weight (X8) was significantly correlated with seed yield per plant (P <0.01). Stepwise regression and path analyses indicated that only pod number per plant and 1000-grain weight contributed significantly to seed yield per plant at P <0.01 and P <0.05, respectively. The regression formula for contributions of pod number per plant (X7) and 1000-grain weight (X8) to seed yield per plant (Y) is Y = 0.006 X7 + 1.222 X8 - 7.191. The path coefficient of pod number per plant to seed yield per plant was 0.967 and that of 1000-grain weight was 0.194. The determination coefficient of pod number per plant and 1000-grain weight to seed yield per plant was 0.983 and the determination coefficient of other agronomic traits was 0.130. Coefficient of variance indicated that the length of primary inflorescence showed the greatest variation, followed by seed yield per plant, pod number per plant, number of secondary branches, branching height, pod number of primary inflorescence, number of first branches, seed yield per plot, 1000-grain weight and plant height. It was suggested that seed yield per plant in Crambe might be improved by increasing the pod number per plant through selection or cultivation, but the negative correlation between pod number per plant and 1000-grain weight also needs to be considere

Isolation and characterization of the omega-6 fatty acid desaturase (FAD2) gene family in the allohexaploid oil seed crop Crambe abyssinica Hochst

Crambe (Crambe abyssinica Hochst ex. R. E. Fries) is an ideal crop for industrial oil production because of its high erucic acid content (C22:1, approx. 60 %) in its seed oil. The value of crambe oil can be improved by increasing C22:1 content or reducing polyunsaturated fatty acids (PUFA). The FAD2 gene plays a critical role in PUFA biosynthesis. To identify targets for breeding, we characterized FAD2 in crambe for copy number and expression profile. Seven copies of FAD2 were detected in allohexaploid crambe. Three genes (CaFAD2-C1, -C2 and -C3) were expressed in multiple tissues, including root, seedling, leaf, flower, bud and developing seeds. In developing seeds, the expression of these genes was upregulated with CaFAD2-C3, being expressed predominantly with a peak at 20 days after pollination. This gene is therefore a promising candidate gene for determining PUFA levels in seed oil. Four other FAD2 genes were considered to be “pseudo-genes” as they harbour internal stop codons and were not expressed. Among the six crambe varieties with consistent variation in oil composition, no nucleotide polymorphisms were found in the CaFAD2-C1, -C2 and -C3 genes. In seeds at 30 days after pollination, statistically significant expression level polymorphisms for only one gene, CaFAD2-C2, was found among the varieties. However, although significantly different, the difference in expression was small and did not explain the variation in oil composition. Given the absence of genetic variation in CaFAD2 genes in crambe breeding lines, breeding for high erucic acid content calls for a molecular breeding approach whereby mutations are chemically induced to increase the genetic variation

Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis

A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487–6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type

Regeneration and transformation of Crambe abyssinica

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 potential of using crambe meristematic tissue for genetic transformation andin vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated

Detection of induced mutations in CaFAD2 genes by next generation sequencing leading to the production of improved oil composition in Crambe abyssinica

Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents