Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary Under an intermediate temperature regime (23° C/18° C; day/night), microsporogenesis in stamens of the ogu cytoplasmic male-sterile (CMS) line of Brassica napus terminated by the tetrad stage, although in some cases degeneration of the sporogenous tissue occurred prior to meiosis. In most cases the tetrads were collapsed and bounded by a sparse exine, but contained many organelles. Also, the tapetum in CMS anthers was abnormal and often highly vacuolated by the tetrad stage. Under low temperature conditions (18° C/15° C; day/night), neither microsporogenous nor tapetal tissues were observed. In the normal stamens, no differences were observed under different temperature regimes. In conjunction with the adjoining paper, this study demonstrates that temperature conditions strongly affect the cytological processes associated with microsporogenesis in the CMS anthers.     

Expression of ogu cytoplasmic male sterility in cybrids of Brassica napus

Summary A light and electron microscopic investigation revealed that ogu cytoplasmic male sterility (CMS) in cybrids of Brassica napus is primarily a deficiency of the tapetum and clearly time and site specific. Three patterns of ogu CMS were found, and specific conclusions drawn. First, the partially male fertile cybrid 23 was highly variable. It sometimes produced heterogeneous stamens with an endothecium formed exclusively around the fertile locules, thus delineating each microsporangium as a functional unit. The second type, including cybrids 27, 58 and 85, on the contrary, was stable and completely male sterile. In the four locules of normal length, microspores were observed to die at the vacuolate polarized stage while the tapetum disappeared prematurely through excessive vacuolization by the end of meiosis followed by a rapid autolysis during the tetrad or early free microspore stage. The subepidermal layer of the locule wall failed to form characteristic thickenings. The male-sterile stamens were completely indehiscent. At the time of anthesis they contained only collapsed empty exines adhering to each other. These cybrids, 27, 58 and 85, were closest to the ogu CMS trait of radish and seemed to be the best suited for further use in plant breeding. The third pattern was found in cybrids 77 and 118, which besides showing abortion of the microsporangia also showed a feminization of the stamens. We suggest that this feminization might be due to an alloplasmic situation associating Brassica napus nuclear genes with the mitochondrial DNA of radish.     

Endogenous Hormones in Seeds, Germination Behaviour and Early Seedling Characteristics in a Normal and ogura Cytoplasmic Male Sterile Line of Rapeseed (Brassica napus L.)

Endogenous hormones, namely cytokinins (CKs), indole-3-aceticacid (IAA) and abscisic acid (ABA) were quantified by specificenzyme-linked immunosorbent assay (ELISA) in the mature seedof normal (cv. Westar) and ogura (ogu) cytoplasmic male sterile(CMS) lines of rapeseed (Brassica napus L.). Dihydrozeatin (DZ)and dihydrozeatin riboside (DZR) were the major CK base andriboside, respectively, in seeds of both the normal and oguCMS lines. The normal seed had more than 4-fold DZ levels incomparison to that of ogu CMS. On the other hand, the ogu CMSseed had higher levels of CK o-glucosides and CK. nucleotidesthan normal seed. Seeds of the normal line contained 5-foldmore IAA but had one-quarter the level of ABA in comparisonto those of the ogu CMS line. The normal line also had greaterseed diameter and weight than the ogu CMS line and the normalseed germinated earlier than the male sterile seed. DZ (10^–6M) promoted the germination of ogu CMS seeds, but it was notcomparable to that of the normal line. ABA (10^–6 M) inhibitedseed germination of ogu CMS but had little effect on the normalline. The normal seedlings had shorter primary roots, more lateralroots, longer hypocotyls, greater cotyledon fresh weight andhigher chlorophyll levels in comparison to ogu CMS seedlings.Exogenously supplied DZ, IAA and ABA affected the various parametersof both the normal and ogu CMS seedlings, but in most casesdid not fully restore the differences in the two lines. The results presented show that in the ogura cytoplasmic malesterile line of B. napus (1) a number of seed and seedling characteristicsare affected, and (2) the altered seed morphology is accompaniedby changes in the levels of various hormones. Key words: Brassica napus, cytoplasmic male sterility, enzyme-linked immunosorbent assay, hormone, seed germination     

High Temperature Induced Male and Female Sterility in Canola (Brassica napus L.)

Male and female sterility was induced in canola or rapeseed(Brassica napus L. cv. Westar) flowers grown under high temperatureconditions (32°C/26°C; day/night). At maturity, themajority of flower buds remained closed but had protruding stigmas.The stamens were reduced in size and the anthers showed abnormalmicrosporogenesis. The gynoecia, although normal in appearance,did not set seed and ovule development was aberrant. Flowersof the ogu CMS line of B. napus also showed similar ovular abnormalitiesunder high temperatures and male fertility was not restoredin them. The observations presented suggest that high temperaturesin the field may adversely affect the yield of canola. Temperature, male and female sterility, rapeseed, canola, Brassica napus     

Comparative analysis of stamen polypeptides of a rapeseed cultivar,Regent, a CMS polima line,and temperature-restored male-fertile polima line

Summary Polypeptides were extracted from stamens of a rapeseed (Brassica napus) cultivar, Regent, a near isogenic male-sterile line, Polima-R7 (Pol-R7), and a high-temperature-restored malefertile Pol-R7 (TR) and subsequently separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis under denaturing conditions. Four variable polypeptides with a pI around 6 were observed. Two stamen polypeptides (40000 Da, 38000 Da) were unique to Regent, and the other two (32000 Da, 30000 Da) were unique to the male-sterile Pol-R7. When the male-sterile Pol-R7 was treated with day/night temperatures of 30°/24° C for 7–10 days prior to flowering, both polypeptides unique to Regent reappeared, while the smaller polypeptides disappeared. Temperature-restored male-fertile Pol-R7 (TR) produced fertile pollen, while its short stamen filaments resembled those of the male-sterile Pol-R7. These changes in protein expression may be causally related to the CMS phenotype.     

Arabidopsis thaliana chromosome III restores fertility in a cytoplasmic male-sterile Brassica napus line with A. thaliana mitochondrial DNA

Somatic Brassica napus (+) Arabidopsis thaliana hybrids with a cytoplasmic male sterility (CMS)-inducing cytoplasm were screened for fertility-restored plants. One line was selected and recurrently backcrossed with the maintainer line, B. napus, resulting in fertile/sterile segregating populations. Restriction fragment length polymorphism mapping showed the co-segregation of A. thaliana chromosome (chr) III markers with the fertility trait. As it was not possible to stabilise the fertility trait via selfings, a dihaploidisation strategy was assessed. Ninety haploid plants were regenerated and analysed with numerous simple sequence length polymorphism (SSLP) markers. Markers covering both arms of A. thaliana chr III were present in two plants, whereas no A. thaliana DNA could be detected in the other plants. Following colchicine-induced chromosome doubling only these two plants with A. thaliana DNA produced fertile offspring. In one of the two lines, however, the A. thaliana-specific DNA markers and fertility were lost in subsequent generations. The other line remained fertile after repeated selfings. Using genomic in situ hybridisation (GISH) we were able to demonstrate that this latter line possessed a disomic addition of the A. thaliana chromosome. The restored line was comparable to the maintainer line with respect to flower morphology, but the petals and stamens were slightly reduced in size. The homeotic conversion of stamens to pistil-like structures, which is typical for the CMS line, was reversed, and stamens with a normal appearance with viable pollen appeared. Flowering time was as in the CMS line—in both lines it was delayed in comparison to the maintainer line. The introgressed chromosome also contributes to several pleiotropic effects, such as reduced leaf crinkling and shorter stems. The ability to restore fertility through the introgression of nuclear genes from the main cytoplasmic donor species indicates that the CMS trait in this system mainly is due to B. napus/A. thaliana alloplasmic incompatibility and not mitochondrial DNA rearrangements. Further exploitation of the material is discussed.Communicated by C. Möllers     

Genetic characterization of a new cytoplasmic male sterility system (<Emphasis Type="Italic">hau</Emphasis>) in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">juncea</Emphasis> and its transfer to <Emphasis Type="Italic">B. napus</Emphasis>

A novel cytoplasmic male sterility (CMS) was identified in Brassica juncea, named as hau CMS (00-6-102A). Subsequently, the male sterility was transferred to B. napus by interspecific hybridization. The hau CMS has stable male sterility. Flowers on the A line are absolutely male sterile, and seeds harvested from the line following pollinations with the maintainer gave rise to 100% sterile progeny. The anthers in CMS plants are replaced by thickened petal-like structures and pollen grains were not detected. In contrast, in other CMS systems viz. pol, nap, tour, and ogu, anthers are formed but do not produce viable pollen. The sterility of hau CMS initiates at the stage of stamen primordium polarization, which is much earlier compared with the other four CMS systems. We have successfully transferred hau CMS from B. juncea to B. napus. Restorer lines for pol, ogu, nap, and tour CMS systems were found to be ineffective to restore fertility in hau CMS. Sixteen out of 40 combinations of mitochondrial probe/enzyme used for RFLP analysis distinguished the hau CMS system from the other four systems. Among these sixteen combinations, five ones alone could distinguish the five CMS systems from each other. The evidence from genetic, morphological, cytological and molecular studies confirmed that the hau CMS system is a novel CMS system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Temperature-induced modifications in the surface features of stamens of a tomato mutant: An SEM study

Summary Stamenless-2 (sl₂/sl₂) is a temperature-sensitive mutant of tomato (Lycopersicon esculentum) which exhibits altered stamen development under different temperatures (Sawhney 1983). By using scanning electron microscopy, this study was conducted to investigate the differentiation of surface features of mutant and normal stamens grown under different temperatures, with the view to further determine the role of temperature in gene expression in stamen development. Mutant stamens grown under intermediate temperatures (23 °C day/18 °C night) differed from the normal in hair production, the shape of epidermal cells and in the pattern of cuticular thickenings. Under low temperatures (18 °C day/15 °C night), all surface features of mutant stamens closely resembled the normal, whereas under high temperatures (28 °C day/23 °C night), the patterns and types of hairs, epidermal cells, stomata, and cuticular thickenings on mutant stamens were similar to that of a gynoecium. The staminal features of normal stamens were not affected by different temperatures. This study shows that the expression of the sl₂/sl₂ allele is influenced by temperature conditions to the extent that the pattern of cellular differentiation characteristic of either the stamens or the carpels can be induced in mutant stamens.     

Synthesis of Brassica oleracea/Brassica napus somatic hybrid plants with novel organeile DNA compositions

Summary Broccoli (Brassica oleracea L. italica) hypocotyl protoplasts were fused with mesophyll protoplasts of two B. napus lines, one carrying the Ogura (ogu) cms cytoplasm, and the other carrying a hybrid cytoplasm consisting of ogu mitochondria combined with triazine-tolerance-conferring chloroplasts from ctr cytoplasm. Two male-sterile somatic hybrids were recovered from the fusion of broccoli protoplasts with those of ogu/ctr cybrid B. napus. The ogu mtDNAs and ctr cpDNAs were not altered in these hybrids. Four male-sterile plants were recovered from the somatic hybridization of broccoli with ogu cms B. napus. Three of these possessed mitochondrial genomes that appeared to have resulted from recombination between the ogu and normal B. oleracea (ole) mtDNAs, while the fourth possessed an unrearranged ogu mtDNA. All four of these plants had B. oleracea cpDNA, and none displayed the seedling chlorosis associated with ogu chloroplasts. Most of the plants recovered from these fusions had the chromosome number expected of B. oleracea + B. napus hybrids (2n = 56). The novel cytoplasms may prove to be useful for the molecular analysis of Brassica cms and for the production of hybrid Brassica.     

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.     

Development of a novel Sinapis arvensis disomic addition line in Brassica napus containing the restorer gene for Nsa CMS and improved resistance to Sclerotinia sclerotiorum and pod shattering

An allo-cytoplasmic male sterile line, which was developed through somatic hybridization between Brassica napus and Sinapis arvensis (thus designated as Nsa CMS line), possesses high potential for hybrid production of rapeseed. In order to select for restorer lines, fertile plants derived from the same somatic hybridization combination were self-pollinated and testcrossed with the parental Nsa CMS line for six generations. A novel disomic alien addition line, B. napus–S. arvensis, has been successfully developed. GISH analysis showed that it contains one pair of chromosomes from S. arvensis and 19 pairs from B. napus, and retains stable and regular mitotic and meiotic processes. The addition line displays very strong restoration ability to Nsa CMS line, high resistance to Sclerotinia sclerotiorum and a low incidence of pod shattering. Because the addition line shares these very important agricultural characters, it is a valuable restorer to Nsa CMS line, and is named NR1 here (Nsa restorer no. 1).     

Changes in protein and amino acid content during anther development in fertile and cytoplasmic male sterile Petunia

Summary Development of anthers in cytoplasmic male sterile (CMS) Petunia diverges from the normal sequence of events early in meiosis. Quantitative and qualitative changes in morphology, proteins and free amino acid contents correlate with this divergence. In anthers of the fertile line (5719), total protein content increases, and SDS-PAGE protein patterns change as the anthers mature. Enhanced levels of three polypeptides with molecular weights of 64,000, 63,000 and 45,000 daltons characterize premeiosis in fertile anthers. Protein levels and patterns from anthers of the CMS line (5707) show little alteration during anther development. Protein synthesis seems to be at least partially blocked in the CMS microspore. The 63,000 and 45,000 dalton proteins are not present, and the absence of any unique protein(s) in the CMS line argues against a virus as the causal agent of CMS in Petunia. Analysis of free amino acids from anthers of the fertile line shows levels of proline and pipecolic acid 2–3 and 10–20 fold higher, respectively, than in the CMS line. The amino acids incorporated into proteins show no such differences; analysis of protein hydrolysates shows similar levels of each amino acid in both fertile and CMS lines at every developmental stage examined.     

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated ‘126-1’, was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F₁s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for ‘126-1’ cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of ‘126-1’ cytoplasm for hybrid seed production. The uniqueness of ‘126-1’ has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.     

Isolation and characterization of temperature-sensitivelethal(2)giant larva alleles

Summary In this paper we present an analysis of the behavior ofl(2)gl ^tsimaginal wing discs during culture in adult hosts. Thel(2)gl ^tslarvae reared at 29° C contain two types of wing discs, those that are morphologically normal and those that are abnormal. When discs of both types are cultured in adult hosts at 29° C, the restrictive temperature, they give rise to transplantable neoplastic tissue. However, when the 29° C reared discs are cultured at 15° C, the permissive temperature, the morphologically normal discs maintain their morphology, but the morphologically abnormal discs give rise to neoplasms. Thel(2)gl ^tslarvae reared at 15° C contain only morphologically normal discs. When these discs are cultured in adult hosts at 29° C they give rise to neoplasms, however if the discs are cultured at 15° C they maintain their normal morphology. These results demonstrate: (1) that all wing imaginal discs obtained from 29° C rearedl(2)gl ^tslarvae are competent to undergo neoplastic development, (2) the morphologically abnormal discs obtained from the 29° C rearedl(2)gl ^tslarvae are committed to neoplastic development, (3) the neoplastic development of the morphologically normal discs is temperature dependent, (4) once the neoplastic development of thel(2)gl ^tsdiscs has been initiated the process is not readily reversible. In addition, the ability ofl(2)gl ^tswing discs to perform epimorphic regulation was tested by amputating morphologically normal permissively rearedl(2)gl ^tswing discs and culturing both fragiments at the permissive temperature. Fragments of control wild-type discs maintained their morphology during culture at the permissive temperature. However, both fragments of txel(2)gl ^tsdiscs became neoplastic. This result is discussed with respect to a possible role for thel(2)gl ⁺function in epimorphic regulation and with respect to the phenomena of tumor promotion in vertebrates.     

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion

Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.     

Host plant selection by the pollen beetle Meligethes aeneus

A simple bioassay is described for the response of Meligethes aeneus F. (Coleoptera: Nitidulidae) to different plants and plant parts. Assays with non-hibernated beetles showed that (1) cruciferous buds and stamens were greatly preferred to petals and leaves; (2) stamens were most preferred when the quantity of mature pollen was at maximum; (3) anthers were preferred to filaments; (4) stamens from five different spring cultivars of Brassica napus were preferred at significantly different degrees; (5) stamens from four B. campestris cultivars were preferred to B. napus cv. Guilliver, but only when the preferences per mm² were compared; (6) stamens from B. carinata and Sinapis alba were preferred to stamens from B. napus and B. campestris; (7) stamens from Compositae (Cirsium arvense, Leontodon nudicaulis, Sonchus arvensis and Taraxacum sp., Vulgaria group) were much less preferred than B. napus stamens (Cruciferae), but more preferred than stamens from Rosa pimpinellifolia (Rosaceae), whereas stamens from Papaver dubium (Papaveraceae) were not preferred to B. napus leaves; (8) chemical stimuli, including odours, were the most important stimuli in the bioassays. the use of equal crude areas for comparisons is briefly discussed.

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Le choix de plantes-hôtes de méligèthe des cruciferes

Résumé Une méthode simple et rapide, est signalée pour l'épreuve de la réponse de Meligethes aeneus vis-à-vis des plantes différentes et des organs différents de plantes. Des épreuves avec des insectes non-hibernés ont indiqué que (1) des étamines et des boutons sont beaucoup plus préférées que les pétales et les feuilles; (2) la préférence des étamines est au maximum en même temps que le nombre de pollens mûrs; (3) des filaments (sans pollens) sont moins préférés que les anthères; (4) les préférences des étamines de cinq variétés différentes de Brassica napus diffèrent significativement; (5) les préférences des étamines de quatre variétés de B. campestris surmontent celle de B. napus var. Gulliver, mais seulement quand les préférences par mm² sont comparées; (6) les préférences des étamines de B. carinata et Sinapsis alba dépassent celles de B. napus et B. campestris; (7) des étamines de Compositae (Cirsium arvense, Leontodon nudicaulis, Sonchus arvensis et Taraxacum sp., group de Vulgaria) sont beaucoup moins préférées que des étamines de B. napus (Cruciferae), et plus préférées que celles de Rosa pimpinellifolia (Rosaceae), tandis que les étamines de Papaver dubium (Papaveraceae) ne sont pas préférées à les feuilles de B. napus; (8) des stimuli chimiques, les odeurs incluses, étaient les stimuli les plus importants dans les épreuves. La nécessité d'appliquer des étendues standardisées dans les comparaisons est brièvement discutée.

     

Pollen selection for low temperature adaptation in tomato

Summary Pollen selection experiments were conducted in tomato to determine the effects of low temperature conditions during pollination on the rate of root elongation of the progeny. Pollen was harvested from an F₁ interspecific hybrid between a high altitude Lycopersicon hirsutum accession and the cultivated tomato L. esculentum. The pollen was applied to stigmas of malesterile L. esculentum plants maintained in growth chambers set at either 12°C/7°C or 24°C/18°C. BC₁ seeds from the low and normal temperature crosses were germinated and root elongation rate was measured at either 9°C or 24°C. At 9°C, the rate of root elongation for progeny of the low temperature crosses was higher than for progeny of crosses at normal temperatures; at 24°C the rate of root elongation was similar for the two crossing treatments. To compare the temperature responses of the two backcross populations we also calculated the relative inhibitory effect of low temperature on the rate of root elongation: the ratio between the rate of root elongation at 9°C to that at 24°C. Root elongation of seedlings from the low temperature crosses was less inhibited by the cold than root elongation for progeny of the normal temperature crosses. These results suggest a relationship between pollen selection at low temperatures and the expression of a sporophytic trait under the same environmental stress.     

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.