Production of haploids and doubled haploids in oil palm

Background: Oil palm is the world’s most productive oil-food crop despite yielding well below its theoretical maximum. This maximum could be approached with the introduction of elite F1 varieties. The development of such elite lines has thus far been prevented by difficulties in generating homozygous parental types for F1 generation. Results: Here we present the first high-throughput screen to identify spontaneously-formed haploid (H) and doubled haploid (DH) palms. We secured over 1,000 Hs and one DH from genetically diverse material and derived further DH/mixoploid palms from Hs using colchicine. We demonstrated viability of pollen from H plants and expect to generate 100% homogeneous F1 seed from intercrosses between DH/mixoploids once they develop female inflorescences. Conclusions: This study has generated genetically diverse H/DH palms from which parental clones can be selected in sufficient numbers to enable the commercial-scale breeding of F1 varieties. The anticipated step increase in productivity may help to relieve pressure to extend palm cultivation, and limit further expansion into biodiverse rainforest.

Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics

Quantitative analysis by mass spectrometry (MS) is a major challenge in proteomics as the correlation between analyte concentration and signal intensity is often poor due to varying ionisation efficiencies in the presence of molecular competitors. However, relative quantitation methods that utilise differential stable isotope labelling and mass spectrometric detection are available. Many drawbacks inherent to chemical labelling methods (ICAT, iTRAQ) can be overcome by metabolic labelling with amino acids containing stable isotopes (e.g. 13C and/or 15N) in methods such as Stable Isotope Labelling with Amino acids in Cell culture (SILAC). SILAC has also been used for labelling of proteins in plant cell cultures (1) but is not suitable for whole plant labelling. Plants are usually autotrophic (fixing carbon from atmospheric CO2) and, thus, labelling with carbon isotopes becomes impractical. In addition, SILAC is expensive. Recently, Arabidopsis cell cultures were labelled with 15N in a medium containing nitrate as sole nitrogen source. This was shown to be suitable for quantifying proteins and nitrogen-containing metabolites from this cell culture (2,3). Labelling whole plants, however, offers the advantage of studying quantitatively the response to stimulation or disease of a whole multicellular organism or multi-organism systems at the molecular level. Furthermore, plant metabolism enables the use of inexpensive labelling media without introducing additional stress to the organism. And finally, hydroponics is ideal to undertake metabolic labelling under extremely well-controlled conditions. We demonstrate the suitability of metabolic 15N hydroponic isotope labelling of entire plants (HILEP) for relative quantitative proteomic analysis by mass spectrometry. To evaluate this methodology, Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress.

Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

Effects of vitamin E and fish oil inclusion in broiler diets on meat fatty acid composition and on the flavour of a composite sample of breast meat

BACKGROUND: Enriching poultry meat with long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) can increase low population intakes of LC n-3 PUFA, but fishy taints can spoil reheated meat. This experiment determined the effect of different amounts of LC n-3 PUFA and vitamin E in the broiler diet on the fatty acid composition and sensory characteristics of the breast meat. Ross 308 broilers (120) were randomly allocated to one of five treatments from 21 to 42 days of age. Diets contained (g kg−1) 0, 9 or 18 LC n-3 PUFA (0LC, 9LC, 18LC), and 100, 150 or 200 mg LD--tocopherol acetate kg−1 (E). The five diets were 0LC100E, 9LC100E, 18LC100E, 18LC150E, 18LC200E, with four pens per diet, except 18LC100E (eight pens). Breast meat was analysed for fatty acids (uncooked) and sensory analysis by R-index (reheated). RESULTS: LC n-3 PUFA content (mg kg−1 meat) was 514 (0LC100E) and 2236 (9LC and 18LC). Compared with 0LC100E, meat from 18LC100E and 18LC150E tasted significantly different, while 23% of panellists detected fishy taints in 9LC100E and 18LC200E. CONCLUSION: Chicken meat can be enriched with nutritionally meaningful amounts of LC n-3 PUFA, but > 100 mg dl--tocopherol acetate kg−1 broiler diet is needed to protect reheated meat from oxidative deterioration. Copyright © 2010 Society of Chemical Industry

Effect of incremental levels of fish oil in the diet on ruminal lipid metabolism in growing steers

Based on the potential benefits to human health, there is interest in developing sustainable nutritional strategies to enhance the concentration of long-chain n-3 fatty acids in ruminant-derived foods. Four Aberdeen Angus steers fitted with rumen and duodenal cannulae were used in a 4 × 4 Latin square experiment with 21 d experimental periods to examine the potential of fish oil (FO) in the diet to enhance the supply of 20 : 5n-3 and 22 : 6n-3 available for absorption in growing cattle. Treatments consisted of total mixed rations based on maize silage fed at a rate of 85 g DM/kg live weight0·75/d containing 0, 8, 16 and 24 g FO/kg diet DM. Supplements of FO reduced linearly (P < 0·01) DM intake and shifted (P < 0·01) rumen fermentation towards propionate at the expense of acetate and butyrate. FO in the diet enhanced linearly (P < 0·05) the flow of trans-16 : 1, trans-18 : 1, trans-18 : 2, 20 : 5n-3 and 22 : 6n-3, and decreased linearly (P < 0·05) 18 : 0 and 18 : 3n-3 at the duodenum. Increases in the flow of trans-18 : 1 were isomer dependent and were determined primarily by higher amounts of trans-11 reaching the duodenum. In conclusion, FO alters ruminal lipid metabolism of growing cattle in a dose-dependent manner consistent with an inhibition of ruminal biohydrogenation, and enhances the amount of long-chain n-3 fatty acids at the duodenum, but the increases are marginal due to extensive biohydrogenation in the rumen.

Plasma ghrelin and oxytomodulin concentrations in lactating dairy cows receiving abomasal soybean oil, corn starch, and casein infusions

The effects of increased postruminal supply of casein, corn starch, and soybean oil on plasma concentrations of the gastrointestinal hormones ghrelin and oxyntomodulin (OXM) were investigated. Four mid-lactation Holstein cows were used in a 4×4 Latin square. Treatments were continuous abomasal infusions (23h/d) for 7 d of water, soybean oil (500g/d), corn starch (1100g/d), or casein (800g/d). Jugular vein plasma was obtained every 30min for 7h on days 1 and 7. Soybean oil and casein infusion decreased preprandial plasma ghrelin concentration by approximately 20% on both d (time-by-treatment P<0.10); however, dry matter intake (DMI) was depressed only after 7 d of oil infusion. Infusion of soybean oil, corn starch, or casein did not change the plasma OXM concentration (P>0.20). The present data indicate that plasma ghrelin concentration is depressed immediately before feeding by the postruminal infusion of soybean oil and casein, but it is not affected during the postprandial period. Plasma ghrelin concentration was not altered (P>0.20), pre- or postfeeding, by increased postruminal supply of corn starch. In addition, plasma OXM concentration did not respond (P>0.20) to postruminal nutrient infusion. In conclusion, a decrease in DMI when fat is infused could be partially explained by the decrease in prefeeding plasma ghrelin concentration, but a decrease in prefeeding plasma ghrelin concentration is not always associated with a decrease in DMI, as observed for the infusion of casein. Plasma OXM concentration was not affected by postruminal infusion of macronutrients.

The effect of feeding modified soyabean oil enriched with C18 : 4 n-3 to broilers on the deposition of n-3 fatty acids in chicken meat

Supplementing broiler diets with conventional vegetable oils has little effect on the long-chain n-3 PUFA (LC n-3 PUFA) content of the meat. The present study investigated the effect on fatty acid composition and sensory characteristics of chicken meat when broilers were fed oil extracted from soyabeans (SDASOY) that had been genetically engineered to produce C18 : 4n-3 (stearidonic acid (SDA), 240 mg/g oil). Three diets were fed to 120 birds (eight replicate pens of five birds) from 15 d to slaughter (41–50 d). Diets were identical apart from the oil added to them (45 and 50 g/kg as fed in the grower and finisher phases, respectively), which was either SDASOY, near-isogenic soya (CON) or fish oil (FISH). The LC n-3 PUFA content of the meat increased in the order CON, SDASOY and FISH. In breast meat with skin, the SDA concentration was 522, 13 and 37 (sem 14·4) mg/100 g meat for SDASOY, CON and FISH, respectively. Equivalent values for C20 : 5n-3 (EPA) were 53, 13 and 140 (sem 8·4); for C22 : 5n-3 (docosapentaenoic acid (DPA)) 65, 15 and 101 (sem 3·5); for C22 : 6n-3 (DHA) 19, 9 and 181 (sem 4·4). Leg meat (with skin) values for SDA were 861, 23 and 68 (sem 30·1); for EPA 87, 9 and 258 (sem 7·5); for DPA 95, 20 and 165 (sem 5·0); for DHA 29, 10 and 278 (sem 8·4). Aroma, taste and aftertaste of freshly cooked breast meat were not affected. Fishy aromas, tastes and aftertastes were associated with LC n-3 PUFA content of the meat, being most noticeable in the FISH leg meat (both freshly cooked and reheated) and in the reheated SDASOY leg meat.

Contribution of apolipoprotein E genotype and docosahexaenoic acid to the LDL-cholesterol response to fish oil

Objectives: To investigate the impact of apolipoprotein E (apoE) genotype on the response of the plasma lipoprotein profile to eicosapentaenoic acid (EPA) versus docosahexaenoic acid (DHA) intervention in humans. Methods and results: 38 healthy normolipidaemic males, prospectively recruited on the basis of apoE genotype (n = 20 E3/E3 and n = 18 E3/E4), completed a double-blind placebo-controlled cross-over trial, consisting of 3 × 4 week intervention arms of either control oil, EPA-rich oil (ERO, 3.3 g EPA/day) or DHA-rich oil (DRO, 3.7 g DHA/day) in random order, separated by 10 week wash-out periods. A significant genotype-independent 28% and 19% reduction in plasma triglycerides in response to ERO and DRO was observed. For total cholesterol (TC), no significant treatment effects were evident; however a significant genotype by treatment interaction emerged (P = 0.045), with a differential response to ERO and DRO in E4 carriers. Although the genotype × treatment interaction for LDL-cholesterol (P = 0.089) did not reach significance, within DRO treatment analysis indicated a 10% increase in LDL (P = 0.029) in E4 carriers with a non-significant 4% reduction in E3/E3 individuals. A genotype-independent increase in LDL mass was observed following DRO intervention (P = 0.018). Competitive uptake studies in HepG2 cells using plasma very low density lipoproteins (VLDL) from the human trial, indicated that following DRO treatment, VLDL2 fractions obtained from E3/E4 individuals resulted in a significant 32% (P = 0.002) reduction in LDL uptake relative to the control. Conclusions: High dose DHA supplementation is associated with increases in total cholesterol in E4 carriers, which appears to be due to an increase in LDL-C and may in part negate the cardioprotective action of DHA in this population subgroup.

Olive oil increases the number of triacylglycerol-rich chylomicron particles compared with other oils: an effect retained when a second standard meal is fed

Background: Compared with the postprandial events after a single meal, different events occur when a second meal is ingested 4–6 h after a first meal. There is a rapid appearance of chylomicrons in the circulation carrying fat ingested with the first meal, with a peak 1 h after the second meal. Objective: Our goal was to examine whether different dietary oils have effects on the storage of triacylglycerol as a result of differences in their digestion, absorption, and incorporation into chylomicrons. Design: A single-blind, randomized, within-subject crossover design was used to study the effects of palm oil, safflower oil, a mixture of fish and safflower oil, and olive oil on postprandial apolipoprotein (apo) B-48, retinyl ester, and triacylglycerol in the Sf > 400 fraction with the use of a sequential meal protocol. Results: For triacylglycerol, retinyl ester, and apo B-48, the time to reach peak concentration was significantly earlier after the second meal than after the first meal (P < 0.005). This was apparent with each of the dietary oils. The pattern of the apo B-48 response differed significantly among the dietary oils, with olive oil resulting in higher concentrations after both meals (P = 0.003). The ratio of triacylglycerol to apo B-48 was significantly lower after olive oil feeding than after feeding with the other oils (P = 0.02). Conclusions: The rapid entry of chylomicrons after the ingestion of a second meal 5 h after a first meal was seen with all of the oils investigated. The short-term ingestion of olive oil produced more chylomicrons than did the other dietary oils, which may have been due to differences in the metabolic handling of olive oil within the gut.