Cytoplasmic male sterility in rapeseed (Brassica napus L.)

Summary Restriction patterns of chloroplast (cp) and mitochondrial (mt) DNA in Brassica napus rapeseed reveal the alloplasmic nature of cytoplasmic male sterility in this crop. Both the Shiga and Bronowski systems probably exploit cytoplasmic diversity in B. napus cultivars arising from introgression of cytoplasm from the other rapeseed species, B. campestris. Nuclear genes specific to these systems do not cause sterility in maintainers (Bronowski and Isuzu-natane) because they have a campestris cytoplasm, but give rise to sterility in napus cytoplasms. In the course of hybridization to napus cultivars a line with the triazine resistant cytoplasm (a campestris cytoplasm) has undergone an alteration in the mt genome rendering its restriction pattern more similar than previously to that of napus. The alteration may be an inversion between 7.2 and 3.4 kb in length.     

Phytochemical,antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves

The phytochemical, antioxidant and mineral composition of hydroalcoholic extract of leaves of Cichorium intybus L., was determined. The leaves were found to possess comparatively higher values of total flavonoids, total phenolic acids. The phytochemical screening confirmed the presence of tannins, saponins, flavonoids, in the leaves of the plant. The leaf extract was found to show comparatively low value of IC₅₀ for 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition. The IC₅₀ value of chicory leaves extract was found to be 67.2 ± 2.6 μg/ml. The extracts were found to contain high amount of mineral elements especially Mg and Zn. Due to good phytochemical and antioxidant composition, C. intybus L., leaves would be an important candidate in pharmaceutical formulations and play an important role in improving the human health by participating in the antioxidant defense system against free radical generation.     

Large scale purification of rapeseed proteins (Brassica napus L.)

Rapeseed (Brassica napus L.) cruciferin (12S globulin), napin (2S albumin) and lipid transfer proteins (LTP) were purified at a multi-g scale. The procedure developed was simple, rather fast and resolutive; it permitted the recovery of these proteins with a good yield, such as 40% for cruciferin and 18% for napin. Nanofiltration eliminated the major phenolic compounds. The remaining protein fraction was fractionated by cation exchange chromatography (CEC) on a streamline SP-XL column in alkaline conditions. The unbound neutral cruciferin was polished by size exclusion chromatography. The alkaline napin isoforms and LTP, adsorbed on the beads, were eluted as a whole fraction and further separated by an other CEC step at acidic pH. Napins were polished by hydrophobic interaction chromatography (HIC). The fractions were characterized by reverse phase HPLC, electrophoresis, N-terminal sequencing and mass spectrometry. All the fractions contained less than 5% of impurities.     

Association mapping of six yield-related traits in rapeseed (Brassica napus L.)

Yield is one of the most important traits for rapeseed (Brassica napus L.) breeding, but its genetic basis remains largely ambiguous. Association mapping has provided a robust approach to understand the genetic basis of complex agronomic traits in crops. In this study, a panel of 192 inbred lines of B. napus from all over the world was genotyped using 451 single-locus microsatellite markers and 740 amplified fragment length polymorphism markers. Six yield-related traits of these inbred lines were investigated in three consecutive years with three replications, and genome-wide association studies were conducted for these six traits. Using the model controlling both population structure and relative kinship (Q + K), a total of 43 associations (P < 0.001) were detected using the means of the six yield-related traits across 3 years, with two to fourteen markers associated with individual traits. Among these, 18 markers were repeatedly detected in at least 2 years, and 12 markers were located within or close to QTLs identified in previous studies. Six markers commonly associated with correlated traits. Conditional association analysis indicated that five of the associations between markers and correlated traits are caused by one QTL with pleiotropic effects, and the remaining association is caused by linked but independent QTLs. The combination of favorable alleles of multiple associated markers significantly enhances trait performance, illustrating a great potential of utilization of the associations in rapeseed breeding programs.     

Thermal reactivity of rapeseed (Brassica napus L.) under different gas atmospheres

Non-isothermal thermogravimetric method was applied to rapeseed to investigate its thermal reactivity under both individual dynamic atmospheres of nitrogen, steam, carbon dioxide and dry air, and under several mixtures of these gases (nitrogen+steam), (steam+dry air), and (nitrogen+carbon dioxide+steam). In this method, the sample was heated from ambient to 1273 K with a constant heating rate of 20 K/min. The trend of the TGA curves and the derived DTG profiles were interpreted regarding the conversion yields and the reactivity of the samples. Conversion yields of biomass to gaseous products changed in the range of 75-94% on original basis, with respect to the atmosphere under which the experiment was carried out. The maximum rates of the mass losses from the sample were found between 2.6 and 4.3 mg/min. The activation energies were calculated using the method of Coats-Redfern, and varied between 21.3 and 96.0 kJ/mol.     

Embryogenesis following cryopreservation in isolated microspores of rapeseed (Brassica napus L.)

A simple procedure is described for cryopreservation of isolated microspores of rapeseed in liquid nitrogen without loss of embryogenic capacity (i.e. embryogenes is can still be induced following freezing). Microspores frozen in Lichter's (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichter's medium with 13% sucrose, and supplemented with 0.5 M glycerol and 0.5 M DMSO produced no embryos. Regeneration of embryos obtained from frozen microspores yielded 88% diploid and 12% haploid plants, while embryos from unfrozen controls produced 7% diploids and 93% haploids. The potential to increase the efficiency of the rapeseed haploidy system using cryopreservation is discussed in light of these results.     

Effect of genotype on shoot regeneration from cotyledonary explants of rapeseed (Brassica napus L.)

Summary Ability of shoot regeneration from cotyledonary explants of rapeseed (B. napus) was surveyed for 100 cultivars. Effects of explant age and plant growth regulators were determined before screening the genotypes. The optimal condition required 4-day-old cotyledons as explant and 4.0 mg/l benzylaminopurine as plant growth regulator. Gas-permeable tape as sealing material was more effective for shoot regeneration than Parafilm. When testing cultivars, shoot regeneration response was strongly influenced by genotype with a range of variation from 97% (percentage of explants regenerating shoots) in Arabella and Norin 26 to 0% in Norin 18 and Norin 30. The response was not dependent on origin and cropping type (spring vs. winter type). The ability of shoot regeneration was not related to that of microspore embryogenesis. The regenerants rooted on medium containing 2.0 mg/l indole-3-butyric acid and after planting in soil flowered and set seeds. Histological studies showed that shoot meristems developed in callus which had grown from the vascular bundle tissue within 8 days.Abbreviations BA 6-benzylaminopurine - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid     

Subunit composition of the globulin fraction of rapeseed (Brassica napus L.)

Multidimensional gel electrophoretic procedures have been employed to studythe polypeptide composition of the 12 S globulin (cruciferin) from rapeseed (Brassica napus L. cv. Tandem). Cruciferin was shown to consist of three main groups of proteins with M_r in the range of 60-52 (A group), 37-35 and 31-29 (respectively B₁ and B₂ groups) and 24-22 (C group). When reduced the A protein group gave rise to two classes of polypeptide constituents. The larger had M_r and isoelectric points (6.4–7.1) corresponding to those of B protein groups whereas the smaller were essentially composed of M_r 22 and a specific 25 kilodaltons (kD) polypeptide with basic isoelectric points. The initial B and C protein groups were unaltered by reducing conditions. These results support the notion that native cruciferin is composed of subunits with large and small polypeptides linked by disulphide bonds and of similar or closely-related polypeptides which are not covalently bonded.     

Phytochemical composition and in vitro biological activities of Morinda citrifolia fruit juice

Morinda citrifolia is a plant with broad nutraceutical and therapeutic effects and used in the traditional treatment of several ailments. The objective of this work is to investigate the phytochemistry of the fruit juice of M. citrifolia on one hand and on other hand to evaluate its antiradical and antibacterial activity. The phytochemical investigation was carried out by tube staining tests of the extract of two types of fruit juice of M. citrifolia. The antioxidant activity of these juices was evaluated by reducing the DPPH radical method. Concerning the antibacterial activity, it was tested on the in vitro growth of 10 reference bacterial strains using the well diffusion method. Qualitative phytochemistry of M. citrifolia fruit juices revealed the presence of large groups of secondary metabolites including polyphenols, reducing compounds, mucilage and terpernoids. The antioxidant activity of M. citrifolia fruit juices is dose-dependent and higher than that of ascorbic acid. Antimicrobial activity on other hand revealed that fruit juices inhibit growth inhibitory activity of Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, S. epidermidis, Proteus vulgaris, Streptococcus oralis, Enterococcus faecalis and Escherichia coli. This observed difference is significant for each juices on the strains (p < 0.001). These results support the use of M. citrifolia in traditional medicine and are the starting points for the development of a new drug to combat both dietary conditions and chronic conditions associated with oxidative stress.     

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent K_M was 6.5 mmol l^-1, with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l^-1 NaCl and on the presence of at least 0.1 mol l^-1 NaCl in the test mixture. Desoxycholate and up to 0.1 mol l^-1 CaCl₂ also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.     

Evidence of cross-reactivity between porcine pancreatic and rapeseed (Brassica napus L.) lipases

A cross-reactivity between animal and plant lipases was determined, using immunological techniques. It was shown by ELISA and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Pre-incubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. Antibodies were able to precipitate lipase activity from 3-day-old rapeseed crude extract. These epitopes seem to be located in the catalytic site, suggesting that a consensus sequence exists in oleaginous lipases and that it will be universal.     

Fatty acid inheritance in microspore-derived Populations of spring rapeseed (Brassica napus L.)

Summary Hard seededness in soybean [Glycine max (L.) Merr.] is a quantitative trait that affects the germination rate, viability, and quality of stored seeds. We have used 72 restriction fragment length polymorphisms (RFLPs) to identify genomic regions containing quantitative trait loci (QTL) affecting hard seededness in a segregating population from a G. max by a Glycine soja (Sieb. & Zucc.) cross. Five independent RFLP markers were found to be associated with variation in the hard-seeded trait. These markers and the epistatic interactions between them explain 71% of the variation for hard seededness. A genomic region associated with the i locus accounted for 32% of the variation in this segregating population. This study illustrates one approach to physiological genetic studies in plants.Journal Paper No. J-13557 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2763     

Discrimination among cultivars of rapeseed (Brassica napus L.) using DNA polymorphisms amplified from arbitrary primers

RAPDs (Randomly Amplified Polymorphic DNAs) were used to discriminate among 23 cultivars of oilseed rape (Brassica napus) selected from several breeding programs. A set of 100 random sequence 10-mer primers were tested, of which 70 produced bands and 22 showed evidence of polymorphism. A selection of six primers produced 23 polymorphic bands of between 300 to 2200 base pairs in size, sufficient to distinguish between the cultivars. An analysis of seed of five cultivars obtained from four different sites showed stability of banding pattern over source of seed. The analysis was repeated using four different thermocyclers, each of which produced the same band pattern. UPGMA cluster analysis indicates that the relationships among some of the cultivars is closer for those from the same breeding program than for those from different programs. The results of this study show that RAPDs can be used as a method of identification for oilseed rape cultivars.Contribution number 941     

Antibacterial and antioxidant activities of flavonoids from Lannea alata (Engl.) Engl. (Anacardiaceae)

Two new prenylated flavonoids, lanneaflavonol (1) and dihydrolanneaflavonol (2) together with the known compounds myricetin-3-O-α-rhamnopyranoside (myricitrin) (3) and myricetin-3-O-α-arabinofuranoside (betmidin) (4), lupeol (5) and sitosterol (6) were isolated from the roots of Lannea alata. Compounds 1–4 exhibited good antibacterial and radical scavenging activity with the glycosides 3 and 4 showing better antioxidant activity than the aglycones 1 and 2 and myricetin-3-O-α-arabinofuranoside (4) showing the best antimicrobial activity followed by the aglycones 1 and 2. Betmidin (4) with an arabinose moiety at the 3-O-position showed the best antibacterial activity against Gram-positive bacteria, followed by the prenylated dihydroflavonol (2), whilst the prenylated linear flavonol (1) showed limited activity against Gram-negative bacteria. The arabinofuranoside (4) followed by the rhamnopyranoside (3) showed the best antioxidant activity comparable to that of ascorbic acid. The biological activities justify the ethnomedicinal uses of the plant in the management of diseases associated with Gram-positive bacteria, such as being used to treat injuries and wounds.     

Synthesis of high erucic acid rapeseed (Brassica napus L.) somatic hybrids with improved agronomic characters

Novel Brassica napus somatic hybrids have been created through protoplast fusion of B. oleracea var. botrytis and B. rapa var. oleifera genotypes selected for high erucic acid (22:1) content in the seed oil. Fifty amphidiploids (aacc) and one putative hexaploid (aacccc) hybrid were recovered in one fusion experiment. Conversely, only one amphidiploid and numerous regenerates with higher DNA contents were produced in a similar fusion using a different B. rapa partner. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Analysis of organellar DNA revealed a distinct bias toward the inheritance of chloroplasts from the B. rapa (aa) genome. All amphidiploids set self-pollinated seed. A erucic acid content as high as 57.4% was found in the seed oil of one regenerated plant. Fatty acid composition was stable in the R₁ generation and was coupled with increased female fertility. Other novel agronomic characters in the hybrids recovered include large seed size, lodging resistance, and non-shattering seed pods.     

Agravitropic behaviour of roots of rapeseed (Brassica napus L.) transformed by Agrobacterium rhizogenes

Transgenic hairy roots of Brassica napus (cv. Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots. The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes. The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia. Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected. The transformed nature of the roots was confirmed by Southern blot analyses. The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings. While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground. The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy. At the ultrastructural level no major differences could be detected between the roots studied. There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type. Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.     

Flavonol glycosides from the leaves of Boldoa purpurascens and their anti-inflammatory properties

Boldoa purpurascens is used in Latin America and the Caribbean as a potent diurectic. Phytochemical analysis has shown the presence of flavonoids and other active compounds. In the present work, three flavonol glycosides were isolated from the leaves of the plant. Their structures have been determined by mass spectrometry and by 1D and 2D NMR analysis as 6-methoxykaempferol-3-O-[α-l-rhamnopyranosyl-(1”’ → 2”)]-β-d-xylopyranoside (1); 3,4′,5-trihydroxy-6,7-methylenedioxyflavone-3-O-[α-l-rhamnopyranosyl-(1”’ → 2”)]-β-d-glucopyranoside (2); and 3,4′,5′,5-tetrahydroxy-6,7-methylenedioxyflavone-3-O-[α-l-rhamnopyranosyl-(1”’ → 2”)]-β-d-xylopyranoside (3). Compounds 1 and 3 are reported for the first time from nature. The NF-κB luciferase assay showed that these compounds have a partial inhibitory effect on NF-κB activation, compound 2 being the most potent one. In the carrageenan induced paw oedema assay in rats, the flavonoid fraction showed acute anti-inflammatory activity, with the highest percentage of inhibition (75.8%) at a dose of 40 mg/kg.     

Molecular analysis and expression of a floral organ-specific polygalacturonase gene isolated from rapeseed (Brassica napus L.)

High throughput screening of stage-specific differentially expressed genes in a Brassica napus two-line Rs1046A/B subtractive library was used to identify the BnQRT3 gene associated with cell wall metabolism. Phylogenetic analysis indicates the protein product of BnQRT3 is polygalacturonase. According to cytological comparisons of Rs1046 sterile and fertile anthers, RT–PCR studies and in situ hybridizations, BnQRT3 is expressed most strongly in floral organs and may play an essential role in pollen maturation. Analysis of the histological staining pattern of BnQRT3 promoter-GUS constructs in transgenic Arabidopsis and Brassica napus revealed that proximal part of 5′-flanking region directed expression in the vascular tissue of filaments, veins in sepal and petals, stigma, branch connective and the floral organ abscission zone during the open flower stage. In the meanwhile, Activity of BnQRT3 was detected in the anthers, which commences at the microsporocyte stage and persists as anther approaches dehiscence. Strong GUS expression also can be observed in the vascular tissue of leaves and stem by compression with forceps or excision, suggesting that the BnQRT3 promoter is responsive to wounding.