Weicong Qi

Dedicated Industrial Oilseed Crops as Metabolic Engineering Platforms for Sustainable Industrial Feedstock Production

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.

Functional analysis of the omega-6 fatty acid desaturase (CaFAD2) gene family of the oil seed crop Crambe abyssinica

BackgroundCrambe abyssinica produces high erucic acid (C22:1, 55-60%) in the seed oil, which can be further increased by reduction of polyunsaturated fatty acid (PUFA) levels. The omega-6 fatty acid desaturase enzyme (FAD2) is known to be involved in PUFA biosynthesis. In crambe, three CaFAD2 genes, CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3 are expressed.ResultsThe individual effect of each CaFAD2 gene on oil composition was investigated through studying transgenic lines (CaFAD2-RNAi) for differential expression levels in relation to the composition of seed-oil. Six first generation transgenic plants (T1) showed C18:1 increase (by 6% to 10.5%) and PUFA reduction (by 8.6% to 10.2%). The silencing effect in these T1-plants ranged from the moderate silencing (40% to 50% reduction) of all three CaFAD2 genes to strong silencing (95% reduction) of CaFAD2-C3 alone. The progeny of two T1-plants (WG4-4 and WG19-6) was further analysed. Four or five transgene insertions are characterized in the progeny (T2) of WG19-6 in contrast to a single insertion in the T2 progeny of WG4-4. For the individual T2-plants of both families (WG19-6 and WG4-4), seed-specific silencing of CaFAD2-C1 and CaFAD2-C2 was observed in several individual T2-plants but, on average in both families, the level of silencing of these genes was not significant. A significant reduction in expression level (P < 0.01) in both families was only observed for CaFAD2-C3 together with significantly different C18:1 and PUFA levels in oil.ConclusionsCaFAD2-C3 expression is highly correlated to levels of C18:1 (r = -0.78) and PUFA (r = 0.75), which suggests that CaFAD2-C3 is the most important one for changing the oil composition of crambe.

Regeneration and transformation of Crambe abyssinica

BackgroundCrambe 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.ResultsDifferent 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.ConclusionsPresent research revealed the potential of using crambe meristematic tissue for genetic transformation and in 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.

Erratum: Screening for recombinants of Crambe abyssinica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

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.

Genomic and transcriptomic sequencing of Rosa hybrida provides microsatellite markers for breeding, flower trait improvement and taxonomy studies

BackgroundRosa hybrida is a valuable ornamental, food and medicinal crop worldwide, but with relatively limited molecular marker resources, especially for flower-specific markers. In this study, we performed genomic and floral transcriptomic sequencing of modern rose. We obtained comprehensive nucleotide information, from which numerous potential simple sequence repeat (SSR) markers were identified but were found to have high rates of amplification failure and PCR product redundancy.ResultsWe applied a filtering strategy for BLAST analysis with the assembled genomic sequence and identified 124,591 genomic and 2,292 EST markers with unique annealing sites. These markers had much greater reliability than those obtained before filtering. Additional BLAST analysis against the transcriptomic sequences uncovered 5225 genomic SSRs associated with 4100 transcripts, 2138 of which were associated with functional genes that were annotated against the non-redundant database. More than 90% of these newly developed molecular markers were polymorphic, based on PCR using a subset of SSRs to analyze tetraploid modern rose accessions, diploid Rosa species and one strawberry accession. The relationships among Rosa species determined by cluster analysis (based on these results) were in agreement with modern rose breeding history, whereas strawberry was isolated in a separate cluster, as expected.ConclusionsOur results provide valuable molecular-genetic tools for rose flower trait improvement, breeding and taxonomy. Importantly, we describe a reproducible organ-specific strategy for molecular marker development and selection in plants, which can be applied to other crops.