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.     

The canola microspore-derived embryo as a model system to study developmental processes in plants

The ability of microspores to undergo embryo development after a successful induction treatment provides a unique experimental system to study a variety of developmental processes in plants. Recent published results focus on the cellular and molecular aspects of the early induction process. In this review, besides summarizing the current findings, the advantages of using the MDE system to study other aspects of embryo development are emphasized. The continual improvement of culturing procedures, media components, and molecular methods guarantees exciting new findings in the near future.     

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     

Bioassembly of acyl lipids in microspore-derived embryos of Brassica campestris L.

The native lipid composition and the capacity of cell-free extracts to biosynthesize acyl lipids in vitro were determined for the first time using the recently reported microspore-derived (MD) embryo system from the Brassica campestris low erucic acid line BC-2 (Baillie et al. 1992). The total lipid fraction isolated from midcotyledonary stage MD embryos (21 days in culture) was composed primarily of triacylglycerol (76%) with an acyl composition quite similar to that of mature BC-2 seed. When incubated in the presence of glycerol-3-phosphate, ¹⁴C 181-CoA, and reducing equivalents, homogenates prepared from 21-day cultured MD embryos were able to biosynthesize glycerolipids via the Kennedy pathway. The maximum in vitro rate of triacylglycerol biosynthesis could more than account for the known rate of lipid accumulation in vivo. The homogenate catalyzed the desaturation of 181 to 182 and to a lesser extent, 183. The newly-synthesized polyunsaturated fatty acids initially accumulated in the polar lipid fraction (primarily phosphatidic acid and phosphatidylcholine) but began to appear in the triacylglycerol fraction after longer incubation periods. As expected for a low erucic acid cultivar, homogenates of MD embryos from the BC-2 line were incapable of biosynthesizing very long chain monounsaturated fatty acyl moieties (201 and 221) from 181-CoA in vitro. Nonetheless, embryo extracts were still capable of incorporating these fatty acyl moieties into triacylglycerols when supplied with ¹⁴C 201-CoA or ¹⁴C 221-CoA. Collectively, the data suggest that developing BC-2 MD embryos constitute an excellent experimental system for studying pathways for glycerolipid bioassembly and the manipulation of this process in B. campestris.Abbreviations CPT sn-1,2-diacylglycerol cholinephosphotransferase - DAG diacylglycerol - DGAT diacylglycerol acyltransferase - DGDG digalactosyldiacylglycerol - G-3-P glycerol-3-phosphate - G-3-PAT glycerol-3-phosphate acyltransferase - LPA lyso-phosphatidic acid - LPAT lyso-phosphatidic acid acyltransferase - LPC lyso-phosphatidylcholine - LPCAT acyl-CoA: lyso-phosphatidylcholine acyltransferase - LPE lyso-phosphatidylethanolamine - MGDG monogalactosyldiacylglycerol - PA phosphatidic acid - PA Phosphatase, phosphatidic acid phosphatase - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - TAG triacylglycerol - 181-CoA oleoyl-Coenzyme A - 181 oleic acid, cis-9-octadecenoic acid - 182 linoleic acid, cis-9,12-octadecadienoic acid - 183 -linolenic acid, cis-9,12,15-octadecatrienoic acid - 201 cis-11-eicosenoic acid - 221 erucic acid, cis-13-docosenoic acid; all other fatty acids are designated by number of carbon atoms: number of double bonds National Research Council of Canada Publication No. 35896     

Cellular changes during heat shock induction and embryo development of cultured microspores ofBrassica napus cv. Topas

Summary Brassica napus cv. Topas microspores, isolated and cultured near the time of the first pollen mitosis and subjected to a heat treatment of 24 h, can be induced to develop into haploid embryos. This is a study of microspore structure during induction and embryo determination. Early during the 32.5 °C incubation period the nucleus moved away from the edge of the cell, and granules, 30 to 60 nm in diameter, appeared in the mitochondria and as a cluster in the cytoplasm. Cells divided symmetrically and at the end of the heat treatment, acquired the features of induced bicellular structures described previously. The features persisted as the cells divided randomly within the exine for 4–7 days following heat induction. Multicellular structures released from the exine underwent periclinal divisions resulting in protoderm differentiation of the globular embryo, thus determining embryo development. The cytoplasm of early heart-stage embryos contains abundant polyribosomes. Non-embryogenic development was indicated by large accumulations of starch and/or lipid and thickened cell walls or an unorganized pattern of cell division following release of the multicellular structures from the exine. Embryogenesis is discussed in terms of induction, embryo determination and development.     

Induction of embryogenesis with colchicine instead of heat in microspores ofBrassica napus L. cv. Topas

Prior to this report, heat treatment (32.5°C, 24 h) was the method used to induce embryogenesis fromBrassica napus microspores. Continuous culture at 25°C results in pollen development. This study shows that colchicine alone, at the non-inductive temperature of 25°C, can induce embryogenesis, thus demonstrating that heat shock is not required for embryogenic induction inB. napus cv. Topas. Embryogenic frequencies of over 15% were obtained by culturing isolated microspores with 25 M colchicine for 42 h at 25°C. The microspore developmental stages responsive to colchicine were unicellular vacuolate and late unicellular, somewhat earlier stages than the population responsive to heat induction. Other groups have reported that heat-shock proteins are essential to the induction of embryogenesis. The present study offers a method of embryogenic induction without the use of heat which will allow discrimination between the factors associated with response to heat shock and those involved with changing cell development.Abbreviations LU Late-unicellular - PPB Preprophase band - UV unicellular-vacuolate The authors wish to thank C. Bornman for his interest and encouragement. We gratefully acknowledge support from the School of Graduate Studies and Research, Queen's University to J.-P. Z., from Hilleshog AB, Sweden to D.H.S., and from the Natural Sciences and Engineering Research Council of Canada to D.H.S. and W.N. Plant Research Centre contribution No. 1595.     

Correlations between gametophytic (pollen) and sporophytic (seed) generations for polyunsaturated fatty acids in oilseed rape Brassica napus L.

Summary Lipids were extracted from the diploid seed and haploid pollen of Brassica napus L. Two fractions of pollen lipids, namely the diploid-specified pollen-coat and the haploid-specified internal cytoplasmic lipids were obtained. Significant correlations exist between pollen and seed generations for linoleic (182) and linolenic (183) acids. In pollen internal storage lipids, the level of 183 is positively correlated and the level of 182 is negatively correlated with the level of 183 in seed lipids. Evidence is presented that strongly supports the hypothesis that lipid biosynthesis occurs within the pollen and that synthesis is specified by haploid genes. These data support the concept of pollen selection, so that selecting among living pollen grains for superior individuals has potential as a new plant breeding tool for improving seed oil quality.     

The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L.

Developing cocoa cotyledons accumulate initially an unsaturated oil which is particularly rich in oleate and linoleate. However, as maturation proceeds, the characteristic high stearate levels appear in the storage triacylglycerols. In the early stages of maturation, tissue slices of developing cotyledons (105 days post anthesis, dpa) readily accumulate radioactivity from [¹⁴C]acetate into the diacylglycerols and label predominantly palmitate and oleate. In older tissues (130 dpa), by contrast, the triacylglycerols are extensively labelled and, at the same time, there is an increase in the percentage labelling of stearate. Thus, the synthesis of triacylglycerol and the production of stearate are co-ordinated during development. The relative labelling of the phospholipids (particularly phosphatidylcholine) was rather low at both stages of development which contrasts with oil seeds that accumulate a polyunsaturated oil (e.g. safflower). Microsomal membrane preparations from the developing cotyledons readily utilised an equimolar [¹⁴C]acyl-CoA substrate (consisting of palmitate, stearate and oleate) and glycerol 3-phosphate to form phosphatidate, diacylglycerol and triacylglycerol. Analysis of the [¹⁴C]acyl constituents at the sn-1 and sn-2 positions of phosphatidate and diacylglycerol revealed that the first acylase enzyme (glycerol 3-phosphate acyltransferase) selectively utilised palmitate over stearate and excluded oleate, whereas the second acylase (lysophosphatidate acyltransferase) was highly selective for the unsaturated acyl-CoA. On the other hand, the third acylase (diacylglycerol acyltransferase) exhibited an almost equal selectivity for palmitate and stearate. Thus, stearate is preferentially enriched at position sn-3 of triacylglycerol at 120–130 dpa because of the relatively higher selectivity of the diacylglycerol acyltransferase for this fatty acid compared with those of the other two acylation enzymes.Abbreviation dpa days post anthesis We are grateful to Drs. G. Pettipher (Cadbury-Schweppes, Reading, UK), M. End and P. Hadley (Department of Horticulture, University of Reading) for the supply of cocoa pods and to the Agricultural and Food Research Council for financial support. We also wish to thank Dr. S. Stymne (Swedish University of Agricultural Sciences, Uppsala, Sweden) for a generous gift of acyl-CoA substrates.     

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     

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.     

Developmental regulation of aldoxime formation in seedlings and mature plants of Chinese cabbage (Brassica campestris ssp. pekinensis) and oilseed rape (Brassica napus): Glucosinolate and IAA biosynthetic enzymes

The first steps in the biosynthesis of glucosinolates and indole-3-acetic acid (IAA) in oilseed rape (Brassica napus L.) and Chinese cabbage (Brassica campestris ssp. pekinensis) involve the formation of aldoximes. In rape the formation of aldoximes from chain-extended amino acids, for aromatic and aliphatic glucosinolate biosynthesis, is catalysed by microsomal flavin-containing monooxygenases. The formation of indole-3-aldoxime from l-tryptophan, the potential precursor of both indole-3-acetic acid and indolyl-glucosinolates, is catalysed by several microsomal peroxidases. The biosynthesis of glucosinolates and indole-3-acetic acid was shown to be under developmental control in oilseed rape and Chinese cabbage. No monooxygenase activities were detected in cotyledons or old leaves of either species. The highest monooxygenase activities were found in young expanding leaves; as the leaves reached full expansion and matured the activities decreased rapidly. The indole-aldoxime-forming activity was found in all of the tissues analysed, but there was also a clear decrease in foliar activity with maturity in leaves of rape and Chinese cabbage. Partial characterisation of the Chinese cabbage monooxygenases showed that they have essentially identical properties to the previously characterised rape enzymes; they are not cytochrome P450-type enzymes, but resemble flavin-containing monooxygenases. No monooxygenase inhibitors were detected in microsomes prepared from either cotyledons or old leaves.Abbreviations DHMet dihomomethionine - FMO flavin-containing monooxygenase - HPhe homophenylalanine - IAA indole-3-acetic acid - l-Phe l-phenylalanine - l-Trp l-tryptophan - MO monooxygenase - IAALD indole-3-acetaldehyde - IAOX indole-3-aldoxime - THMet trihomomethionine     

Changes in synthesis and localization of members of the 70-kDa class of heat-shock proteins accompany the induction of embryogenesis inBrassica napus L. microspores

Elevation of the culture temperature to 32°C for approximately 8 h can irreversibly change the developmental fate of isolatedBrassica napus microspores from pollen development to embryogenesis. This stress treatment was accompanied by de-novo synthesis of a number of heat-shock proteins (HSPs) of the 70-kDa class: HSP68 and HSP70. A detailed biochemical and cytological analysis was performed of the HSP68 and HSP70 isoforms. Eight HSP68 isoforms, one of which was induced three fold by the stress treatment, were detected on two-dimensional immunoblots. Immunocytochemistry revealed a co-distribution of HSP68 with DNA-containing organelles, presumably mitochondria. Six HSP70 isoforms were detected, one of which was induced six fold under embryogenic culture conditions. During normal pollen development, HSP70 was localized in the nucleoplasm during the S phase of the cell cycle, and predominantly in the cytoplasm during the remainder. Induction of embryogenic development in late unicellular microspores was accompanied by an intense anti-HSP70 labeling of the nucleoplasm during an elongated S phase. In early bicellular pollen the nucleus of the vegetative cell, which normally does not divide and never expresses HSP70, showed intense labeling of the nucleoplasm with anti-HSP70 after 8 h of culture under embryogenic conditions. These results demonstrate a strong correlation between the phase of the cell cycle, the nuclear localization of HSP70 and the induction of embryogenesis. As temperature stress alone is responsible for the induction of embryogenic development, and causes an altered pattern of cell division, there might be a direct involvement of HSP70 in this process.Abbreviations HSP heat-shock protein - 2-D two-dimensional - DAPI 4,6-diamidino-2-phenylindole. 1-D = one-dimensional - pI isoelectric point     

Study of microspore-culture responsiveness in oilseed rape (Brassica napus L.) by comparative mapping of a F2 population and two microspore-derived populations

RFLP segregation analyses were performed on a F₂ population and two F₁ microspore-derived populations from the same cross between a microspore culture-responsive parent (Topas) and a non-responsive parent (Westar). A total of 145 loci were detected with 87 cDNA clones. Eighty-two markers were common across all three populations. A total of 66 markers was assembled into 18 linkage groups and 16 markers remained unlinked. Segregation distortions were significant for 29% of the markers in the F₂ population and 23% and 31% in microspore-derived populations M3 and M5, respectively. An equivalent number of markers showed biased segregation towards each parental allele in the F₂ population while more markers showed a significant deviation from the expected Mendelian ratio towards the responsive parent in both microspore-derived populations. Different subsets of markers showed segregation distortions in the three populations indicating that the selective pressures leading to microsporederived plants are different from those acting during selfing of the F₁. Linkage groups 1 and 18 were identified as putative chromosomal regions associated with microspore-culture responsiveness.     

Selection for salt tolerance in vitro using microspore-derived embryos ofBrassica napus cv. topas,and the characterization of putative tolerant plants

Microspore-derived embryos ofBrassica napus cv. Topas that survived salt stress, were obtained after selection against otherwise lethal doses (0.6 and 0.7%) of NaCl after mutagen treatment. A total of 10 salt-surviving embryos were obtained out of a possible 834 000 embryos that were mutagenized. One embryo out of a possible 845 000 obtained from nonmutagenized controls survived but failed to develop into a plant. Visual assessment after salt stress indicated that both the putative salt-tolerant plants and plants from control seeds behaved similarly. However, based on individual characteristics related to salt tolerance, one of the lines (PST-2) accumulated less sodium and retained more potassium, and hence was able to maintain a more favorable Na:K ratio as compared to the controls under salt stress. Also chlorophylla fluorescence induction and quenching signals indicated a high energetic state of the thylakoid membranes in PST-2 under salt stress. The other putative salt-tolerant line (PST-1) had a higher background level of proline that may have enabled it to survive salt stress during initial screening, although its later performance was no better than the control plants.     

Isozyme analysis as a tool for introgression of Sinapis alba germ plasm into Brassica napus

Summary Isozyme analysis of Brassica napus cv Topas and CGRC5006 as well as of Sinapis alba cv Emergo revealed significant polymorphism between the two species for the isozymes, aconitate hydratase, glucose phosphate isomerase, and diaphorase. F₁ hybrids between B. napus 5006 and S. alba cv Emergo were backcrossed to B. napus cv Topas, and the S₁ progeny of the first two backcrosses were studied isozymically. At the backcross one level the frequency of S. alba or S. alba plus B. napus patterns observed ranged from 18% to 87% across the four lines studied. There were differences between lines for the frequency of S. alba patterns, which could have an impact on the efficiency of selection for subsequent backcrossing. By the backcross two generation in one of the two lines studied, GR86-24, the S. alba patterns for GPI and DIA had been lost, while in the other line, GR86-28, the S. alba pattern for ACO had been lost, resulting in lost opportunity for S. alba gene transfer. In a wide cross such as S. alba x B. napus, which requires an intensive effort to accomplish, the isozymes ACO, GPI, and DIA may serve as useful markers to ensure gene transfer between the two species has occurred. In addition, the identification of lines with divergent isozyme patterns from B. napus will provide the basis for establishing linkages between S. alba traits of interest and isozyme markers.     

Novel seed lipid phenotypes in combinations of mutants altered in fatty acid biosynthesis inArabidopsis

Summary The seed fatty acid (FA) composition of various single mutant combinations ofArabidopsis thaliana that affect FA biosynthesis has been examined. Double mutant combinations offae, a mutation affecting CIS elongation, and a series of four other FA biosynthetic mutants were synthesized. The four other single mutants were:fad2 andfad3, which are deficient in 181 and 182 desaturation, respectively;fab1, which is elevated in 160 and decreased in 181; andfab2, which is elevated in 180 and decreased in 181. The superimposition of two blocks in the FA biosynthetic pathway leads to dramatic changes in the FA content of the double mutants. The tenArabidopsis stocks analyzed to date (wild-type, five single mutants, and four double mutants) make seed oils with a wide range of FA compositions, and illustrate the diversity of oils it is possible to obtain from a single plant species.     

Cell cycle dependent distribution of phosphorylated proteins in microspores and pollen ofBrassica napus L., detected by the monoclonal antibody MPM-2

Summary The monoclonal antibody MPM-2, which interacts with a mitosis-specific phosphorylated epitope, has been used to study phosphorylation of proteins in microspores and pollen ofBrassica napus. One- (1-D) and two-dimensional (2-D) immunoblots revealed that MPM-2 recognized a family of phosphorylated proteins in freshly isolated microspores and pollen. The same set of phosphorylated proteins was found after 8 h of culture at embryogenie (32 °C) and non-embryogenic (18 °C) conditions. Two major spots were observed on 2-D immunoblots, one of which (M_r75 kDa, pI5.1) co-localized with the 70 kDa heat shock protein. Immunolabelling of sectioned microspores and pollen showed that MPM-2 reactive epitopes were predominantly observed in the nucleoplasm from G₁ until G2-phase, and in the cytoplasm during mitosis. This may be due to a cell cycle related translocation of phosphoproteins from the nucleus to the cytoplasm, or alternate phosphorylation and dephosphorylation in nucleus and cytoplasm. Detectability of epitopes on sections depended on the embedding procedure. Cryo processing revealed epitope reactivity in all stages of the cell cycle whereas polyethylene glycol embedded material showed no labelling in the cytoplasm during mitosis. Processing might reduce the antigenicity of cytoplasmic MPM-2 detectable proteins, probably due to dephosphorylation. The MPM-2 detectable epitope was observed in all cells investigated, irrespective of culture conditions, and its intracellular distribution depended on the cell cycle stage and was not related to the developmental fate of the microspores and pollen.     

A comparison of anther and microspore culture as a breeding tool in Brassica napus

Summary A direct comparison of microspore culture and anther culture was made in Brassica napus using F₁ crosses of Regent (canola) by Golden (rapeseed), and their reciprocals, as well as a hybrid between Reston and a highly embryogenic, canola-quality breeding line (G231) as donor plants. The study confirmed that microspore culture can be ten times more efficient than anther culture for embryo production. Embryo yields from cultures initiated from the Reston x G231 were four-fold greater than those initiated from the Regent x Golden crosses, and significant differences were also detected among cultures initiated from the different Regent x Golden crosses. These results illustrate the influence that donor plant genotype has on embryo production. However, superior embryogenic potential among donor material was not always coincident with superior plant production. The average haploid-todiploid ratio in microspore-derived regenerates was 21 for the population obtained from the Regent x Golden crosses but 11 for the Reston x G231 cross. For both types of material, the frequency of diploids increased upon repeated cycles of explanting. A field study showed that there were no differences between the populations of anther-derived and microspore-derived spontaneous diploid and doubled haploid lines, with respect to the days required for them to flower or to mature. The information is valuable for canola breeding programs considering the use of haploidy.