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PubMed2000

Determination of glucosinolates in canola seeds using anion exchange membrane extraction combined with the high-pressure liquid chromatography detection.

A rapid, simple, and reliable method for the determination of individual glucosinolates in canola seeds was developed using a semiquantitative extraction of glucosinolates with anion exchange membranes and HPLC detection. In this one-step extraction procedure, a membrane (7 cm(2)) is placed in the seed suspension prepared by grinding and boiling 0.8 g of seeds in 20 mL of water. After 10 min of shaking on the mechanical shaker, the membrane is removed from the suspension, washed, and transferred to a vial containing 5 mL of 1 N tetramethylammonium chloride. The glucosinolates are eluted from the membrane by shaking the membrane for 10 min with the eluting solvent. The glucosinolate content in membrane eluates is determined by HPLC using sinigrin standards. A coefficient of variation ranging from 1.9 to 7.6% for aliphatic glucosinolates indicated very good reproducibility of the method. Because of the instability of 4-hydroxyglucobrassicin, the coefficient of variation for the determination of this indolyl glucosinolate was 13.9%. To verify the results of the membrane extraction/HPLC detection, this new method was compared with the existing colorimetric and GC procedures. Very good correlation (R(2) = 0.98) was obtained between the total glucosinolates determined by the membrane extraction/HPLC method and the palladate colorimetric procedure for 17 canola varieties. Concentrations of individual glucosinolates in five canola varieties were compared with the GC data. Very good agreement between these two methods was obtained for aliphatic glucosinolates. However, the membrane extraction/HPLC method yielded slightly higher values for 4-hydroxyglucobrassicin than the GC method, possibly indicating that the decomposition of this glucosinolate was reduced during the sample extraction with the membranes. The simplicity and low cost of the membrane extraction/HPLC method make it an attractive alternative to the existing procedures for glucosinolate analysis in canola seeds.

Chromatography, High Pressure Liquid↗
PubMed2004

Three-dimensional seed reconstruction from an incomplete data set for prostate brachytherapy.

Intra-operative dosimetry in prostate brachytherapy requires 3D coordinates of the implanted, radioactive seeds. Since CT is not readily available during the implant operation, projection x-rays are commonly used for intra-operative seed localization. Three x-ray projections are usually used. The requirement of the current seed reconstruction algorithms is that the seeds must be identified on all three projections. However, in practice this is often difficult to accomplish due to the problem of heavily clustered and overlapping seeds. We have developed an algorithm that permits seed reconstruction from an incomplete data set. Instead of all three projections, the new algorithm requires only one of the three projections to be complete. Furthermore, even if all three projections are incomplete, it can reconstruct 100% of the implanted seeds depending on how the undetected seeds are distributed among the projections. The method utilizes the principles of epipolar imaging geometry and pseudo-matching of the undetected seeds. The algorithm was successfully applied to a large number of clinical cases where seeds imperceptibly overlap in some projections.

Algorithms↗
PubMed2006

Improved resistance to controlled deterioration in transgenic seeds.

We show that seed-specific overexpression of the sunflower (Helianthus annuus) HaHSFA9 heat stress transcription factor (HSF) in tobacco (Nicotiana tabacum) enhances the accumulation of heat shock proteins (HSPs). Among these proteins were HSP101 and a subset of the small HSPs, including proteins that accumulate only during embryogenesis in the absence of thermal stress. Levels of late embryogenesis abundant proteins or seed oligosaccharides, however, were not affected. In the transgenic seeds, a high basal thermotolerance persisted during the early hours of imbibition. Transgenic seeds also showed significantly improved resistance to controlled deterioration in a stable and transgene-dependent manner. Furthermore, overexpression of HaHSFA9 did not have detrimental effects on plant growth or development, including seed morphology and total seed yield. Our results agree with previous work tentatively associating HSP gene expression with phenotypes important for seed longevity. These findings might have implications for improving seed longevity in economically important crops.

DNA-Binding Proteins↗
PubMed1993

Isolation and characterization of wheat triticin cDNA revealing a unique lysine-rich repetitive domain.

Polyclonal antibodies were raised against a purified 22 kDa triticin polypeptide (delta) and were used to screen a wheat seed cDNA library in the Escherichia coli expression vector lambda gt11. The isolated cDNA clones were grouped into three families based on their cross-hybridization reactions in DNA dot-blot studies. Southern blots of genomic DNAs extracted from ditelocentric and nullisomic-tetrasomic lines of Chinese Spring wheat, probes with the excised cDNA inserts, indicated that one of the three families (9 clones) had triticin clones. This was finally confirmed by comparing the predicted amino acid sequences of two of these clones (lambda Tri-12, lambda Tri-25) with the published tryptic peptide sequences of triticin. The Southern blots also showed that there is at least one triticin gene located on the short arm of each of the homoeologous group 1 chromosomes (1A, 1B, 1D), although till now no triticin protein product has been identified for the chromosome 1B. The nucleotide sequence of the largest triticin cDNA clone lambda Tri-25 (1567 bp) is presented here, and its predicted amino acid sequence shows strong homology with the legumin-like proteins of oats (12S globulin), rice (glutelin) and legume seeds. A unique feature of the triticin sequence is that it contains a lysine-rich repetitive domain, inserted in the hypervariable region of the typical legumin-like genes. Northern blotting of total RNA extracted from different stages of the developing wheat seed revealed that the triticin gene expression is switched on 5-10 days after anthesis (DAA). There was a steady increase in the level of triticin mRNA until 20 DAA, after which it started decreasing. The maximum mRNA accumulation occurred between 17 and 20 DAA. These observations conform closely with the published data on triticin protein accumulation during grain development.

Amino Acid Sequence↗
PubMed1999

Vacuolar H(+)-ATPase is expressed in response to gibberellin during tomato seed germination.

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA(4+7). Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V(0) membrane sector of vacuolar H(+)-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V(1) sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.

Amino Acid Sequence↗
PubMed2000

Study of phospholipases D and C in maturing and germinating seeds of Brassica napus.

Different forms of phospholipase D (dependent on and independent of the presence of phosphatidylinositol 4,5-bisphosphate, PIP(2)) were identified in maturing and germinating seeds of Brassica napus. Both forms were present in cytosolic and membrane fractions of maturing seeds. PIP(2)-dependent activity increased continuously during seed germination, while PIP(2)-independent activity appeared mostly at the very beginning of seed maturation. PIP(2)-dependent activity was detected mainly in the plasma-membrane fraction. Phosphatidylinositol-specific phospholipase C (PI-PLC) was found only in membrane fractions of both types of developing rape seed tissues. The increasing activities of PLC and PIP(2)-dependent PLD were mainly detected in hypocotyls of seedlings. Some biochemical characteristics of both described enzymes are also presented.

Brassica↗
PubMed2003

An X-ray micro-tomography system optimised for the low-dose study of living organisms.

An X-ray micro-tomography system has been designed that is dedicated to the low-dose imaging of radiation sensitive living organisms and has been used to image the early development of the first few days of plant development immediately after germination. The system is based on third-generation X-ray micro-tomography system and consists of an X-ray tube, two-dimensional X-ray detector and a mechanical sample manipulation stage. The X-ray source is a 50kVp X-ray tube with a silver target with a filter to centre the X-ray spectrum on 22keV.A 100mm diameter X-ray image intensifier (XRII) is used to collect the two-dimensional projection images. The rotation tomography table incorporates a linear translation mechanism to eliminate ring artefact that is commonly associated with third-generation tomography systems. Developing maize seeds (Triticum aestivum) have been imaged using the system with a cubic voxel linear dimension of 100 microm, over a diameter of 25mm and the root lengths and volumes measured. The X-ray dose to the plants was also assessed and found to have no effect on the plant root development.

Equipment Design↗
PubMed2004

Synthesis and evaluation of scaffolds prepared from chitosan fibers for potential use in cartilage tissue engineering.

Tissue engineering concepts and methodologies that employ biocompatible matrices or scaffolds have the potential to meet needs encountered in the repair of defects in articular cartilage. A desirable design parameter in the tissue engineering of cartilage in vitro is the development of seeded scaffolds with appropriate structure, composition, mechanical properties and durability that are similar to normal articular cartilage. Previous methods that have used freeze drying and lyophilization techniques to make foams and hydrogels have not met the scaffold characteristics (porosity, compressive elastic modulus, permeability and viscoelastic properties), which are required of scaffolds slated for use in cartilage tissue engineering applications. Thus there is an impetus to design and develop biomimetic scaffolds that mimic the native ECM of articular cartilage, and distribute strain in a bioresponsive manner to signal seeded chondrocytes to synthesize and organize ECM to result in material properties that are in range of natural cartilage. We have employed the method of electrospinning to prepare scaffolds with oriented and random fiber alignment.

Animals↗
PubMed1988

The sequence of a gene encoding convicilin from pea (Pisum sativum L.) shows that convicilin differs from vicilin by an insertion near the N-terminus.

The sequence of a gene encoding convicilin, a seed storage protein in pea (Pisum sativum L.), is reported. This gene, designated cvcA, is one of a sub-family of two active genes. The transcription start of cvcA was mapped. Convicilin genes are expressed in developing pea seed cotyledons, with maximum levels of the corresponding mRNA species present at 16-18 days after flowering. The gene sequence shows that convicilin is similar to vicilin, but differs by the insertion of a 121-amino-acid sequence near the N-terminus of the protein. This inserted sequence is very hydrophilic and has a high proportion of charged and acidic residues; it is of a similar amino acid composition to the sequences found near the C-terminal of the alpha-subunit in pea legumin genes, but is not directly homologous with them. Comparison of this sequence with the 'inserted' sequence in soya-bean (Glycine max) conglycinin (a homologous vicilin-type protein) suggests that the two insertions were independent events. The 5' flanking sequence of the gene contains several putative regulatory elements, besides a consensus promoter sequence.

Amino Acid Sequence↗
PubMed2001

Effects of dietary maritime pine seed oil on lipoprotein metabolism and atherosclerosis development in mice expressing human apolipoprotein B.

BACKGROUND: Conifer seeds are used for food preparation in several countries. Aim of the study To assess the lipid-lowering and antiatherogenic properties of maritime pine (Pinuspinaster) seed oil. METHODS: The effects of maritime pine oil supplementation (20% w/w) for 2 weeks were compared to those of coconut and sunflower oil in mice expressing human apolipoprotein B (hApoB). Atherosclerosis lesion development was measured in hApoB mice fed 1.25% (w/w) cholesterol and 0.05% (w/w) sodium cholate and either coconut, sunflower or maritime pine oil (20% w/w) for 8 weeks. RESULTS: After 2 weeks of dietary treatment, plasma cholesterol (p < 0.0001), triglyceride (p < 0.0003), phospholipid (p < 0.0001) and apolipoprotein B (p < 0.0001) levels were lower in mice supplemented with maritime pine oil than in those treated with coconut oil. These effects were accounted for by a lowering of LDL-cholesterol, LDL-phospholipids and LDL-triglycerides, as well as a decrease in HDL-cholesterol and HDL-phospholipids. After 8 weeks of dietary treatment cholesterol and cholate, the mean area of aortic lesions was not statistically different between fat groups. CONCLUSIONS: Feeding maritime pine oil is associated with major changes of lipid and lipoprotein levels in hApoB mice. However, in the long term, maritime pine oil has no preventive effect on cholesterol-induced aortic lesion development in hApoB mice.

Animals↗
PubMed2000

HPLC analysis of white lupin isoflavonoids.

An investigation of the HPLC analytical conditions for simple isoflavones, prenylated isoflavones and some of their glucosyl derivatives resulted in reasonable separation and total elution in 35 min when using a reversed-phase C18 Lichrospher column and a gradient elution system of MeCN-THF-H2O. This method was successfully applied to quantify the changes in isoflavonoid constituents in white lupin (Lupinus albus L.) tissues: (a) young legumes (pods and seeds) during maturation, and (b) soaked, germinating seeds. In developing legumes, genistein and 2'-hydroxygenistein, as well as their prenylated derivatives, were present in the pods as the major components, together with minor amounts of glucosides, whereas only minute amounts of isoflavonoids were detectable in the ripening seeds. When soaked with water, mature lupin seeds which normally contain trace amounts of isoflavonoids, started rapidly to biosynthesize simple isoflavones and accumulate large amounts of genistein 7-O-glucoside and its 6"-O-malonyl derivative. These dynamic changes are discussed in relation to the role of isoflavonoids in the lupin defense system.

Chromatography, High Pressure Liquid↗
PubMed2001

Effect of bacterial population density on germination wheat seeds and dynamics of simple artificial ecosystems.

Effect of the size of rhizospheric bacterial populations on germination of seeds and development of simple terrestrial "wheat plants--rhizospheric microorganisms--artificial soil" and "wheat plants-artificial soil" systems has been studied. Experiments demonstrated that within specify ranges in the inoculate, the rhizospheric bacteria are capable of increasing the yield of germinated seeds and stimulate the growth of plantlets. Germination of seeds inoculated with bacteria was either stimulated, or inhibited or remained at control levels depending on the amount of bacteria. Plant biomass growth and total photoassimilation has been found to depend on the amount of bacteria on the plant roots: the higher the amount of bacteria on plant roots, the smaller is the biomass of plants but the total photoassimilation is, higher. Thus, depending on the amount of bacteria on the roots of plants the system either increases the biomass of plants or increases the total photoassimilation, i.e. "pumps" carbon through itself involving bacteria. Grant numbers: N99-04-96017, N15.

Ecosystem↗
PubMed2002

PCR-based assay for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens in bean seeds.

AIMS: To develop a PCR-based protocol for the rapid, sensitive and specific detection of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) in bean seeds. METHODS AND RESULTS: A pair of PCR primers (CffFOR2-CffREV4), targeting the sequence of a cloned DNA fragment of 550 bp amplified in Repetitive-sequence-based-PCR (Rep-PCR) experiments, were designed and shown to specifically amplify a 306-bp DNA fragment using Cff DNA as template. Moreover, this PCR protocol was demonstrated to successfully detect Cff in naturally infected bean seeds in 36 h. CONCLUSIONS: A specific, highly sensitive and rapid PCR assay for the detection of Cff was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: Cff is a seed-borne bacterium on the EPPO A2 quarantine list; this procedure may be useful for routine diagnosis of Cff, overcoming the problems of conventional techniques.

Actinomycetales↗
PubMed2001

Identification of a major gene and RAPD markers for yellow seed coat colour in Brassica napus.

The development of yellow-seeded Brassica napus for improving the canola-meal quality characteristics of lower fibre content and higher protein content has been restricted because no yellow-seeded forms of B. napus exist, and their conventional development requires interspecific introgression of yellow seed coat colour genes from related species. A doubled-haploid (DH) population derived from the F1 generation of the cross 'Apollo' (black-seeded) x YN90-1016 (yellow-seeded) B. napus was analysed via bulked segregant analysis to identify molecular markers associated with the yellow-seed trait in B. napus for future implementation in marker-assisted breeding. A single major gene (pigment 1) flanked by eight RAPD markers was identified co-segregating with the yellow seed coat colour trait in the population. This gene explained over 72% of the phenotypic variation in seed coat colour. Further analysis of the yellow-seeded portion of this DH population revealed two additional genes favouring 'Apollo' alleles, explaining 11 and 8.5%, respectively, of the yellow seed coat colour variation. The data suggested that there is a dominant, epistatic interaction between the pigment I locus and the two additional genes. The potential of the markers to be implemented in plant breeding for the yellow-seed trait in B. napus is discussed.

Brassica napus↗
PubMed2003

Micronutrient fortification of plants through plant breeding: can it improve nutrition in man at low cost?

Can commonly-eaten food staple crops be developed that fortify their seeds with essential minerals and vitamins? Can farmers be induced to grow such varieties? If so, would this result in a marked improvement in human nutrition at a lower cost than existing nutrition interventions? An interdisciplinary international effort is underway to breed for mineral- and vitamin-dense varieties of rice, wheat, maize, beans and cassava for release to farmers in developing countries. The biofortification strategy seeks to take advantage of the consistent daily consumption of large amounts of food staples by all family members, including women and children as they are most at risk for micronutrient malnutrition. As a consequence of the predominance of food staples in the diets of the poor, this strategy implicitly targets low-income households. After the one-time investment is made to develop seeds that fortify themselves, recurrent costs are low and germplasm may be shared internationally. It is this multiplier aspect of plant breeding across time and distance that makes it so cost-effective. Once in place, the biofortified crop system is highly sustainable. Nutritionally-improved varieties will continue to be grown and consumed year after year, even if government attention and international funding for micronutrient issues fades. Biofortification provides a truly feasible means of reaching malnourished populations in relatively remote rural areas, delivering naturally-fortified foods to population groups with limited access to commercially-marketed fortified foods that are more readily available in urban areas. Biofortification and commercial fortification are, therefore, highly complementary. Breeding for higher trace mineral density in seeds will not incur a yield penalty. Mineral-packed seeds sell themselves to farmers because, as recent research has shown, these trace minerals are essential in helping plants resist disease and other environmental stresses. More seedlings survive and initial growth is more rapid. Ultimately, yields are higher, particularly in trace mineral-'deficient' soils in arid regions.

Biological Availability↗
PubMed2004

Application of RAPD and SCAR markers for purity testing of F1 hybrid seed in chili pepper (Capsicum annuum).

A simpler and better method for purity testing of hybrid pepper seed was developed. The simplest method for extracting genomic DNA, the NaOH method, was chosen. Two RAPD markers identifying male and female parents were also developed, and the PCR products of male- and female-specific RAPD markers were cloned and sequenced. From these sequences, new longer primers were constructed for conversion into SCAR markers. In blind tests the RAPD and SCAR markers were able to reliably detect contaminating exotic seeds. These PCR-based markers are therefore directly applicable for purity testing by seed companies. In addition, the PCR products of the SCAR markers could be identified by direct staining methods such as ethidium bromide and pellet painting without electrophoresis.

Capsicum↗
PubMed1980

Comparison of three methods for determining aflatoxins in sunflower seed meals.

Methods developed for the determination of aflatoxins in peanut products, in cottonseed products, and in ginger were compared for their applicability to sunflower seed meal. The peanut products method was inapplicable. Separation from interferences adequate to obtain a limit of determination for aflatoxin B1 around 1 ng/g and recovery of total added aflatoxins of about 97% could be achieved with either the cottonseed or ginger methods. The cleanest extract and the most rapid operation were obtained with the ginger method.

Aflatoxins↗