Transfer of resistance against Phoma lingam to Brassica napus by asymmetric somatic hybridization combined with toxin selection

Summary Irradiated mesophyll protoplasts from nine different accessions of B. juncea, B. nigra and B. carinata, all resistant to Phoma lingam, were used as gene donors in fusion experiments with hypocotyl protoplasts isolated from B. napus as the recipient. A toxin, sirodesmin PL, was used to select those fusion products in which the resistant gene(s) was present. In the fusion experiments different gene donors, various irradiation dosages and toxin treatments were combined. Symmetric and asymmetric hybrid plants were obtained from the cell cultures with and without toxin selection. Isozymes were used to verify hybrid characters in the symmetric hybrids, whereas two DNA probes were used to identify donor-DNA in the asymmetric hybrids. Resistance to P. lingam was expressed in all symmetric hybrids, and in 19 of 24 toxin-selected asymmetric hybrids, while all the unselected asymmetric hybrids were susceptible.     

Use of the polymerase chain reaction to isolate an S-locus glycoprotein cDNA introgressed from Brassica campestris into B. napus ssp. oleifera.

Summary A self-incompatible canola-quality Brassica napus ssp. oleifera line (W1) was generated by introgressing the S-locus from a self-incompatible B. campestris plant into the Westar cultivar. Using the polymerase chain reaction (PCR) with primers derived from conserved regions in S-locus glycoprotein (SLG) alleles, the central region of the active SLG gene (910) was obtained. The remaining portions of the cDNA for this 910 gene were subsequently cloned using the PCR-rapid amplification of cDNA ends (RACE) procedure. Sequence analysis revealed that the 910 cDNA show a high degree of sequence similarity to SLG alleles associated with Class I self-incompatible lines. The 910 gene was found to be absent in the original self-compatible cv. Westar (B. napus) and segregated with self-incompatibility in a mixed population generated from a cross between self-incompatible W1 and self-compatible Westar. RNA blot analysis indicated that high levels of 910 mRNAs were present in the stigma as buds approached anthesis. Thus, the SLG allele of W1 transferred from B. campestris via backcrosses to a line of cv. Westar has been identified.     

Increase of histidine content in Brassica rapa subsp. oleifera by over-expression of histidine-rich fusion proteins

An approach that enables the increase of the quantity of a specific amino acid in crop plants is reported. Oleosin gene from Arabidopsis thaliana or 30K movement protein gene of Tobacco mosaic virus (TMV; genus Tobamovirus) were cloned under the control of napin or hybrid promoters, and in fusion to synthetic poly-histidine (poly-His) sequences for transformation into spring turnip rape (Brassica rapa subsp. oleifera; synonym to B. campestris). The most stable expression cassettes for the poly-His production prior to the plant transformation were selected by analyzing the protein expression in in vitro translation and in transient plant expression systems using GFP as marker. Expression of the poly-His-constructs in transgenic Brassica rapa plants was analyzed using dot and western blotting and PCR. The constructs were stably expressed in the third generation of the transgenic plant lines. Histidine content was measured from the seeds of the transgenic plants, and some plant lines had more than 20% increase in histidine content compared to wild type. The methodology may be widely applicable to increase the content of any amino acid in crop plants including those encoded by rare codons.     

Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro

Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 g d^-1 during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10^-6 M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 g embryo^-1 d^-1) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.Abbreviations ABA abscisic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium lauryl sulfate     

Induction of microspore embryogenesis in Brassica napus L. is accompanied by specific changes in protein synthesis

Culture temperature determines the developmental fate of isolated microspores from Brassica napus L. At 18°C, tricellular pollen develops, whereas culture at 32°C for 8 h leads to the quantitative and synchronous induction of embryogenesis, and ultimately to the formation of embryos. We investigated the changes in protein synthesis that are associated with this 8-h inductive period by using in-situ [³⁵S]methionine labeling, followed by two-dimensional (2-D) gel electrophoretic analysis of the radiolabeled proteins. Qualitative and quantitative computer analyses of 2-D [³⁵S]methionine protein patterns showed six polypeptides specifically labeled under embryogenic culture conditions. Eighteen polypeptides incorporated [³⁵S]methionine at a statistically significant higher rate under embryogenic culture conditions (32°C) than in the controls (18°C), whereas one protein was preferentially labeled under non-embryogenic culture conditions (18°C). These results indicate that only a limited number of proteins detectable in the 2-D gels of microspore extracts are associated with the early induction of embryogenesis. The reproducible identification of the differentially radiolabeled proteins in the 2-D gels allow the sequencing of representative peptides and the isolation of the corresponding cDNAs. This may lead to the identification and characterization of proteins associated with the very first stages of plant embryogenesis.Abbreviations 2-D two-dimensional We would like to thank Dr. H. Van Steeg (Rijks Instituut voor Milieubeheer (RIVM), Bilthoven, The Netherlands) for use of the PhosphorImager apparatus. This research was carried out as part of the EC-Bridge project Regulation of the inductive phase of microspore embryogenesis and EC-Science project The role of mitotic and cytoskeletal genes in the induction of plant cell division.     

Resistance responses to Phoma lingam of plants regenerated from selected cell and embryogenic cultures of haploid Brassica napus

Summary Resistant plants and plants with reduced susceptibility against the pathogen Phoma lingam could be regenerated from selected callus and embryogenic cultures of haploid rape (Brassica napus) previously treated with mutagens. In the two in vitro selection systems used — absence of fungus growth on the cultures after incubation with parasite spores and resistance to the toxic filtrate — the resistance to the toxin was effective. In addition, some regenerants with increased tolerance were obtained from unselected cultures. Resistance tests on regenerated plants were carried out by inoculation of whole plants in the greenhouse, reproducing as much as possible the infection mechanisms which take place under natural conditions. Preliminary results on resistance of the progeny of single susceptible and tolerant regenerants seem to indicate that the acquired resistances are of a genetic nature.Part of these results was reported in a scientific meeting (Die höhere Pflanzenzelle: Möglichkeiten und Grenzen der Forschung) dedicated to Professor G. Melchers in the Max-Planck-Institut für Zellbiologie (Ladenburg) in January 1981     

Brassica napus pollen oleosins possess a characteristic C-terminal domain

The sequences of Brassica napus L. pollen oleosins have been determined and examined. Contrary to a recent report, inferred primary sequences of pollen oleosins do include a unique C-terminal domain characterised by the presence of a repetitive motif of three alanine and one proline residue (AAAP). This motif appears to be present in all oleosins expressed in pollen, but not in oleosins from other tissues.     

A comparative assessment of purification techniques for mesophyll protoplasts of Brassica napus L.

The effects of three different general purification protocols have been assessed quantitatively using mesophyll protoplasts of Brassica napus. Within the initial sample two distinct sub-populations were determined. The methods used influenced the ratio of the vacuolated to chloroplastic type protoplast sub-populations. Overall recovery rates of the initial sample varied according to the method used from 38% to 27%, but the relative recovery of the sub-populations varied considerably with a purified ratio of between 1.0:0.78 to 1.0:7.0. Size distribution profiles of the initial and purified populations are also presented.     

Heterogeneity of the endoplasmic reticulum with respect to lipid synthesis in developing seeds of Brassica napus L.

Studies of the sub-cellular location of storage triacylglycerol (TAG) synthesis in developing embryos of oilseed rape (Brassica napus L.) show that there is heterogeneity of the endoplasmic reticulum (ER) with respect to the enzymes of lipid synthesis. The enzymes of TAG synthesis were detected in two membrane fractions (equilibrium densities 1.05 and 1.10 g· ml^?1) isolated by sucrose-density-gradient centrifugation of homogenates from developing rape embryos. The synthesis of TAG by the lowdensity membranes has not been reported previously and was found in this study because the sucrose density gradients began at only 10% (w/w) sucrose. The pattern of activity of the enzymes involved in the synthesis of TAG in the higher-density fraction closely matched the marker enzymes for the ER; lyso-phosphatidylcholine acyltransferase and cytidine diphosphate-choline:diacylglycerol cholinephosphotransferase. The activity of the ER marker enzymes in the low-density membrane fraction, however, was very much lower when compared to those involved in the synthesis of TAG. Analysis of the lipids extracted from the low-density fraction revealed it contained about 50 mol% TAG compared with 15 mol% in the bulk ER, which may account for the low density of the membranes in this fraction. The possibility that the low-density membranes were the result of contamination of ER by oil bodies was ruled out by the use of oleosins as a marker for oil bodies. It is suggested that the low-density membranes are derived from a domain of the ER which is involved in the formation and secretion of TAG.     

Immunocytochemical localization of myrosinase in Brassica napus L.

The cytological and intracellular localization of myrosinase (EC 3.2.3.1) has been studied by immunochemical techniques using paraffin-embedded sections of radicles and cotyledons from seeds of Brassica napus L. cv. Niklas. For immunolabelling, sections were sequentially incubated with a monoclonal anti-myrosinase antibody and with peroxidase-and fluorescein-isothiocyanate-conjugated secondary antibodies. Enzyme and fluorescence label was present in typical myrosin cells both in radicles and in cotyledons. With higher magnification, fluorescence label revealed that the intracellular localization of myrosinase was associated with the tonoplast-like membrane surrounding the myrosin grains in the myrosin cells. The results also indicate that a large proportion of the positive myrosin cells are located in the second-outermost cell layer of the peripheral cortex region of the radicles.Abbreviations FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - PBS-T PBS with 0.5% (v/v) Tween-20 (polyoxyethylene sorbitane monolaurate) This work was supported by The Norwegian Research Council for Science and the Humanities. We wish to thank Professor Med. O.A. Haugen, Department of Pathology, University of Trondheim, Norway, for the skilful assistance provided regarding fixation and sectioning.     

Non-zygotic embryos of Brassica napus L. contain embryo-specific storage proteins

The storage-protein content of non-zygotic and zygotic embryos of B. napus was compared, using antibodies to guantitate 12S storage protein in extracts by rocket immunoelectrophoresis. Non-zygotic embryos were induced from microspores in anther culture and on the hypocotyls of zygotic embryos in culture. All embryo-like structures were found to contain 12S storage protein, whereas preculture anthers, anthers from which embryos had been removed, and regenerated shoots did not have detectable 12S storage protein. In zygotic embryos, 12S storage protein was first detected at the cotyledon stage, but microsporic embryos contained storage protein at the globular and heart stages. Storage protein levels in microsporic and hypocotyl embryos were low relative to those in zygotic embryos. The largest microsporic embryo had a storage protein concentration of 13 g mg^-1 fresh weight, almost 10 times lower than a mature zygotic embryo. Thus, although storage proteins are present in both zygotic and non-zygotic embryos, the timing and extent of accumulation differ.     

Novel insect resistance in Brassica napus developed by transformation of chitinase and scorpion toxin genes

Transgenic plants with introduced pest-resistant gene offer an efficient alternative insect control. The novel insect-resistant gene combination, chitinase(chi) and BmkIT(Bmk), containing an insect-specific chitinase gene and a scorpion insect toxin gene was introduced into Brassica napus cultivar via Agrobacterium-mediated transformation. Fifty-seven regenerated plantlets with kanamycin-resistance were obtained. Transgenic plants were verified by Southern blot analysis. Enzyme-linked immunosorbent assay (ELISA) and bioassay of artificial inoculation with diamondback moth (Plutella maculipenis) (DBM) larvae indicated that some of the transgenic plants were high-level expression for both chitinase and scorpion toxin proteins and performed high resistance against the tested pest infestation. The genetic analysis of T₁ progeny confirmed that the inheritance of introduced genes followed the Mendelian rules.     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

Pyruvate-kinase isoenzymes from zygotic and microspore-derived embryos of Brassica napus

Polyclonal antibodies against castor-oil seed cytosolic and leucoplastic pyruvate kinases (PK_c and PK_p, respectively; EC 2.7.1.40) were utilized to examine the subunit compositions and developmental profiles of canola (Brassica napus L. cv. Topas) PK_c and PK_p over 6 d of seed germination and 35 d of culture of microspore-derived embryos. The PK_c from germinating seeds appears to be composed of a single type of 56-kDa subunit, whereas the enzyme from cultured embryos contains equal proportions of immunologically related 57- and 56-kDa subunits. The PK_p was immunologically undetectable in germinating seeds, while the enzyme from cultured embryos consisted of immunologically related 64- and 58-kDa subunits in a ratio of about 12, respectively. The large increase in PK activity that occurs between the second and fourth days of seed gemination is based upon de-novo synthesis of PK_c. Between 7 and 14 d of culture of microspore-derived embryos, the levels of PK_p and PK maximal activity increased approx. 3- and 2.5-fold, respectively. These increases were coincident with an approximately fourfold rise in the in-vivo pyruvate: phosphoenolpyruvate concentration ratio. Conversely, PK_c was not only far less abundant relative to PK_p, but its level remained constant over 35 d of microspore-embryo culture. Developing non-zygotic (microspore-derived) embryos strongly resembled ripening zygotic (seed) embryos in terms of PK specific activity as well as relative amounts and subunit compositions of PK_c and PK_p. The results indicate that the synthesis of PK isoenzymes in B. napus seeds is highly regulated and that this regulation follows a preset developmental program.Abbreviations IgG immunoglobulin G - IU international unit - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PK(s) pyruvate kinase(s) - PK_c cytosolic pyruvate kinase - PK_p plastidic pyruvate kinase - PYR pyruvate Plant Research Centre contribution No. 1374We wish to thank Ms. Kathryn Hovey and Ms. Suzanne Belliveau (Agriculture Canada) for their expert assistance in the culturing and harvesting of microspore-derived embryos of canola. This work was supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada.     

Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.)

Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.     

Mapping of genes affecting linolenic acid content in Brassica rapa ssp. oleifera

F₂ progeny segregating for linolenic acid content were used to identify genes and develop markers for linolenic acid in spring turnip rape (Brassica rapa ssp. oleifera). A candidate gene approach applying the rapeseed fad3 gene and bulked segregant analysis with RAPD markers was used. A total of 27 markers were distributed in three linkage groups which each exhibited a QTL for linolenic acid. Jointly the three QTLs accounted for 73.5% of the variation in linolenic acid level in this population. The fad3 gene was mapped near one QTL controlling 23.5% of the variation. Allele-specific markers were developed for fad3 and can be used for marker-assisted selection in future spring turnip rape breeding programmes.