Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: molecular analysis of the cytoplasm of parental lines and corrected progeny

Summary Cytoplasmic differences between male-fertile and male-sterile Brassica napus as well as Raphanus sativus were investigated. Plastids of the male-fertile B. napus were found to differ from those of male-sterile B. napus and R. sativus with respect to DNA restriction enzyme patterns. Differences between male-fertile and male-sterile B. napus mitochondria were detected not only in the restriction fragment patterns of their DNA, but also at the level of expression by in organello translation of mitochondrial polypeptides.The chlorophyll deficiency obtained upon transferral of the male-sterility-conferring radish cytoplasm to a winter variety of B. napus had been corrected earlier through protoplast fusion. The cytoplasmic composition of the corrected lines was analysed using DNA restriction analysis and in organello translation. The stability of the recombined cytoplasm in the corrected lines was confirmed by analysis of the subsequent seed-derived generation.     

Transfer of the Brassica tournefortii cytoplasm to B. napus for the production of cytoplasmic male sterile B. napus

The donor-recipient fusion method was used to combine the cytoplasm of Brassica lournefortii with the nucleus of B. napus for the production of cytoplasmic male sterile (CMS) plants. X-ray-irradiated mesophyll protoplasts of B. tournefortii were fused with iodoacetamide (lOA)-inactivated hypocotye protoplasts of B. napus . Selective conditions of IOA concentrations and X-ray doses were determined, which resulted in recovery of fusion products and inhibition of further growth of unfused parental cells. In total, 54 plants were obtained from different fusion experiments, of which 25 were verified as cybrids or partial hybrids. Mitochondrial DNA (mtDNA) analyses using 5 mitochondrial gene probes revealed that 20 of the 25 fusion-derived plants had mtDNA either identical, or with varying degrees of similarity, to B. lournefortii . These plants were classified into four groups on the basis of pollen viability and number. Seven plants were categorised as male sterile since they did not produce pollen or had non-viable pollen. Of the male sterile plants, five had a mtDNA pattern identical to B. tournefortii and a nuclear DNA content corresponding to B. napus . The nuclear-mito-chondrial constitution of these plants thus indicates that the combination of B. tournefortii cytoplasm with the B. napus nucleus results in CMS. Furthermore, mtDNA analysis of the two additional male sterile plants which displayed a rearranged mtDNA, revealed that the only mtDNA similarity shared among all male sterile plants was specific for B. tournefortii atp6 pattern. This indicates that the atp6 region of B. tournefortii may be involved in the expression of CMS.     

Expression levels of meristem identity and homeotic genes are modified by nuclear-mitochondrial interactions in alloplasmic male-sterile lines of Brassica napus

Homeotic conversions of anthers were found in cytoplasmic male sterile (CMS) plants of Brassica napus derived from somatic hybrids of B. napus and Arabidopsis thaliana. CMS line flowers displayed petals reduced in size and width and stamens replaced by carpelloid structures. In order to investigate when these developmental aberrations appeared, flower development was analysed histologically, ultrastructurally and molecularly. Disorganized cell divisions were detected in the floral meristems of the CMS lines at stage 4. As CMS is associated with mitochondrial aberrations, ultrastructural analysis of the mitochondria in the floral meristems was performed. Two mitochondrial populations were found in the CMS lines. One type had disrupted cristae, while the other resembled mitochondria typical of B. napus. Furthermore, expression patterns of genes expressed in particular floral whorls were determined. In spite of the aberrant development of the third whorl organs, BnAP3 was expressed as in B. napus during the first six stages of development. However, the levels of BnPI were reduced. At later developmental stages, the expression of both BnAP3 and BnPI was strongly reduced. Interestingly the expression levels of genes responsible for AP3 and PI activation such as LFY, UFO and ASK1 were higher in the CMS lines, which indicates that activation of B-genes in the CMS lines does not occur as in B. napus. Disrupted and dysfunctional mitochondria seem to be one of the first aberrations manifested in CMS which result in a retrograde influence of the expression levels of genes responsible for the second and third whorl organ differentiation.     

Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii

Cytogenetic studies were performed on four male-sterile progenies derived from four different cybrids produced between Brassica napus and B. tournefortii using the donor-recipient protoplast fusion method. The objective of these studies was to characterize the nuclear constitution of the plants. Mitotic investigation revealed that three of the four male-sterile lines had 38 chromosomes, which is equal to that of B. napus. The fourth line, C6, had variable chromosome numbers, ranging from 39 to 42 in different plants. The meiotic behavior in each progeny varied distinctly. Of the plants having 38 chromosomes, fairly high chromosome pairing, on average 18.08 bivalents per cell, was detected at metaphase-I. However, univalents with an average of 1.39 per cell, and very low frequencies of trivalents and/or tetravalents, were also observed in the lines. These results revealed that male-sterile cybrid lines were obtained with 38 chromosomes and a relatively high level of chromosome-pairing ability, indicating their potential for establishing a stable male-sterile rapeseed line. Received:15 December 1998 / Accepted:30 January 1999     

Characterization of the 12S storage protein of Brassica napus (cruciferin): Disulfide bonding between subunits

Cruciferin (12S globulin) is a large, neutral, oligomeric protein synthesized in rapeseed ( Brassica napus ) during the seed development. It is composed of six subunit pairs. Each pair consists of one heavy α chain (30 kDa) and one light β chain (20 kDa). Four different subunit pairs exist. In contrast to earlier studies, our investigations using two-dimensional electrophoresis showed, that the majority of α and β chains of each subunit are disulfide-linked. Analysis of subunit composition of cruciferin hexamers by ion-exchange chromatography suggested that a large array of hexamers exist, composed of mixed combinations of the four subunits.     

Variation in the glucosinolate content of oilseed rape (Brassica napus L.) leaves

The glucosinolate content of oilseed rape {Brassica napus) leaves was monitored over the growth period 30–70 days after planting, and a comparison made between a single-low cultivar (low in erucic acid), Bienvenu, and a double-low cultivar (low in erucic acid and glucosinolate), Cobra. In older, fully-expanded leaves the glucosinolate concentration was very low (< 0.3 μmol/ml tissue water) and did not alter during the course of the experiment. In developing sixth leaves glucosinolate content increased rapidly and reached a maximum concentration (4–5 μmol/ml tissue water) 40 days after planting (6 days after leaf emergence). The concentration then declined, to about 1 μmol/ml after 60 days although the total glucosinolate content in leaves continued to increase until 50 days; much of the reduction in concentration was simply a result of leaf expansion. No major differences were seen between the two varieties in total glucosinolate content or in the individual compounds present. Cv. Cobra developed more quickly than cv. Bienvenu so direct comparison between leaves of the two cultivars was complex. When comparing the glucosinolate content of oilseed rape leaves, between cultivars or between treatments, it is vital to ensure that carefully matched leaves of comparable developmental age are selected.     

Cold-deacclimation of oilseed rape (Brassica napus var. oleifera) in response to fluctuating temperatures and photoperiod

The aim of this work was to establish the role of factors that may trigger elongation growth in the dehardening response, namely temperature during daylight, photoperiod and vernalization. Fully cold-acclimated seedlings of winter (with incomplete vernalization) and spring oilseed rape were subjected to deacclimation under temperatures of 2/12, 12/2, 12/12, 12/20, 20/12 and 20/20 degrees C (day/night) and a 12 h photoperiod. Plants were also deacclimated under photoperiods of 8 and 16 h at constant temperatures of 12 and 20 degrees C. After deacclimation, plants were subjected to reacclimation. Results suggest that the level of growth activity induced during deacclimation affects both the deacclimation rate and the capacity for reacclimation. Deacclimation is fully reversible if it is not accompanied by induction of elongation growth. In such cases the rate of the decrease in freezing tolerance depends on the mean temperature of deacclimation. Deacclimation becomes partially or completely irreversible when it is connected with promotion of elongation growth. The stimuli triggering elongation growth during deacclimation may be the growth-promoting temperature (20 degrees C) during the day and the lack of vernalization blockage of elongation growth. When elongation growth was stimulated by other factors such as long-day treatments, rehardening was also disturbed.     

Photosynthesis in leaves and siliques of winter oilseed rape (Brassica napus L.)

The rate of photosynthesis and its relation to tissue nitrogen content was studied in leaves and siliques of winter oilseed rape (Brassica napus L.) growing under field conditions including three rates of nitrogen application (0, 100 or 200 kg N ha^-1) and two levels of irrigation (rainfed or irrigated at a deficit of 20 mm). The predominant effect of increasing N application under conditions without water deficiency was enhanced expansion of photosynthetically active leaf and silique surfaces, while the rate of photosynthesis per unit leaf or silique surface area was similar in the different N treatments. Thus, oilseed rape did not increase N investment in leaf area expansion before a decline in photosynthetic rate per unit leaf area due to N deficiency could be avoided. Much less photosynthetically active radiation penetrated into high-N canopies than into low-N canopies. The specific leaf area increased markedly in low light conditions, causing leaves in shade to be less dense than leaves exposed to ample light. In both leaves and siliques the photosynthetic rate per unit surface area responded linearly to increasing N content up to about 2 g m^-2, thus showing a constant rate of net CO₂ assimilation per unit increment in N (constant photosynthetic N use efficiency). At higher tissue N contents, photosynthetic rate responded less to changes in N status. Expressed per unit N, light saturated photosynthetic rate was three times higher in leaves than in silique valves, indicating a more efficient photosynthetic N utilization in leaves than in siliques. Nevertheless, from about two weeks after completion of flowering and onwards total net CO₂ fixation in silique valves exceeded that in leaves because siliques received much higher radiation intensities than leaves and because the leaf area declined rapidly during the reproductive phase of growth. Water deficiency in late vegetative and early reproductive growth stages reduced the photosynthetic rate in leaves and, in particular, siliques of medium- and high-N plants, but not of low-N plants.     

甘蓝型油菜Pol CMS育性恢复基因的PCR标记

采用恢、保回交群体和集团混合分析法,筛选了１０４０个１０－ｍｅｒ随机引物,找到了与甘蓝型油菜波里马细胞质雄性不育系（Ｐｏｌ ＣＭＳ）育性恢复基因（Ｒｆｐ）连锁的两个ＲＡＰＤ标记Ｓ１０１９７２０和Ｓ１０３６８１０。它们位于Ｒｆｐ的一侧,与该基因的遗传图距分别为５．８ｃＭ和１２．３ｃＭ。随后,克隆并测序这２个多态性片段,根据其２端序列设计了２对２０～２４－ｍｅｒ的特异引物,它们在１３８株的回交群体中Ｐ     

Drought adaptability of Agrobacterium rhizogenes-induced roots in oilseed rape (Brassica napus var. oleifera)

Abstract. Soil grown oilseed rape ( Brassica napus L. var. oleifera M., cv. Darmor) seedlings at the cotyledon stage (one week old), were inoculated in vivo at the base of the hypocotyl with Agrobacterium rhizogenes harbouring the pRi 15834 plasmid. Resulting adventitious root formation was observable about 2 or 3 weeks after infection. Differential Ri-induced root emergence and subsequent development occurred depending on water conditions and closeness of the wounding site to the soil surface: either thin, hairy roots growing rapidly and plagiotropically at the soil level under humid atmosphere, or hairless and fleshy, slowly growing aerial roots developed. The hairy roots were highly drought susceptible, whereas aerial roots revealed some potential for drought tolerance. Unlike normal roots, none of these Ri-induced roots appeared able to give rise to drought rhizogenesis in plants subjected to progressive drought stress. However, under hardening, achieved through successive and moderate drought stress-rehydration cycles, both types of Ri-induced roots improved drought tolerance and could express the morphogenetic differentiation programme leading to the formation of short, tuberized, drought-adapted, roots. These results, discussed in terms of hormonal imbalance and drought tolerance regulation, suggest that the Ri T-DNA gene expression, responsible for adventitious root induction and growth behaviour, is further regulated through the host plant.     

Oilseed rape (Brassica napus) as a resource for farmland insect pollinators: quantifying floral traits in conventional varieties and breeding systems

Oilseed rape (OSR; Brassica napus L.) is a major crop in temperate regions and provides an important source of nutrition to many of the yield‐enhancing insect flower visitors that consume floral nectar. The manipulation of mechanisms that control various crop plant traits for the benefit of pollinators has been suggested in the bid to increase food security, but little is known about inherent floral trait expression in contemporary OSR varieties or the breeding systems used in OSR breeding programmes. We studied a range of floral traits in glasshouse‐grown, certified conventional varieties of winter OSR to test for variation among and within breeding systems. We measured 24‐h nectar secretion rate, amount, concentration and ratio of nectar sugars per flower, and sizes and number of flowers produced per plant from 24 varieties of OSR representing open‐pollinated (OP), genic male sterility (GMS) hybrid and cytoplasmic male sterility (CMS) hybrid breeding systems. Sugar concentration was consistent among and within the breeding systems; however, GMS hybrids produced more nectar and more sugar per flower than CMS hybrid or OP varieties. With the exception of ratio of fructose/glucose in OP varieties, we found that nectar traits were consistent within all the breeding systems. When scaled, GMS hybrids produced 1.73 times more nectar resource per plant than OP varieties. Nectar production and amount of nectar sugar in OSR plants were independent of number and size of flowers. Our data show that floral traits of glasshouse‐grown OSR differed among breeding systems, suggesting that manipulation and enhancement of nectar rewards for insect flower visitors, including pollinators, could be included in future OSR breeding programmes.     

Barriers to gene flow from oilseed rape (Brassica napus) into populations of Sinapis arvensis

One concern over growing herbicide-tolerant crops is that herbicide-tolerance genes may be transferred into the weeds they are designed to control. Brassica napus (oilseed rape) has a number of wild relatives that cause weed problems and the most widespread of these is Sinapis arvensis (charlock). Sinapis arvensis seed was collected from 102 populations across the UK, within and outside B. napus-growing areas. These populations were tested for sexual compatibility with B. napus and it was found that none of them hybridized readily in the glasshouse. In contrast to previous studies, we have found that hybrids can be formed naturally with S. arvensis as the maternal parent. Six diverse B. napus cultivars (Capricorn, Drakkar, Falcon, Galaxy, Hobson and Regent) were tested for their compatibility with S. arvensis but no cultivar hybridized readily in the glasshouse. We were unable to detect gene transfer from B. napus to S. arvensis in the field, confirming the extremely low probability of hybridization predicted from the glasshouse work.     

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus)

Ogura cytoplasmic male sterility (CMS) occurs naturally in radishand has been introduced into rapeseed (Brassica napus) by protoplastfusion. As with all CMS systems, it involves a constitutivelyexpressed mitochondrial gene which induces male sterility tootherwise hermaphroditic plants (so they become females) anda nuclear gene named restorer of fertility that restores pollenproduction in plants carrying a sterility-inducing cytoplasm.A correlative approach using light and electron microscopy wasapplied to define what stages throughout development were affectedand the subcellular events leading to the abortion of the developingpollen grains upon the expression of the mitochondrial protein.Three central stages of development (tetrad, mid-microsporeand vacuolate microspore) were compared between fertile, restored,and sterile plants. At each stage observed, the pollen in fertileand restored plants had similar cellular structures and organization.The deleterious effect of the sterility protein expression startedas early as the tetrad stage. No typical mitochondria were identifiedin the tapetum at any developmental stage and in the vacuolatemicrospores of the sterile plants. In addition, some strikingultrastructural alterations of the cell's organization werealso observed compared with the normal pattern of development.The results showed that Ogu-INRA CMS was due to premature celldeath events of the tapetal cells, presumably by an autolysisprocess rather than a normal PCD, which impairs pollen developmentat the vacuolate microspore stage, in the absence of functionalmitochondria. Key words: Brassica napus, cell death, light and electron microscopy, mitochondria, plastids, pollen development, Ogu-INRA cytoplasmic male sterility, transgenic-restored plants, tapetum Received 30 September 2007; Revised 11 December 2007 Accepted 20 December 2007     

Modelling and estimating pollen movement in oilseed rape (Brassica napus) at the landscape scale using genetic markers

Understanding patterns of pollen movement at the landscape scale is important for establishing management rules following the release of genetically modified (GM) crops. We use here a mating model adapted to cultivated species to estimate dispersal kernels from the genotypes of the progenies of male-sterile plants positioned at different sampling sites within a 10 x 10-km oilseed rape production area. Half of the pollen clouds sampled by the male-sterile plants originated from uncharacterized pollen sources that could consist of both large volunteer and feral populations, and fields within and outside the study area. The geometric dispersal kernel was the most appropriate to predict pollen movement in the study area. It predicted a much larger proportion of long-distance pollination than previously fitted dispersal kernels. This best-fitting mating model underestimated the level of differentiation among pollen clouds but could predict its spatial structure. The estimation method was validated on simulated genotypic data, and proved to provide good estimates of both the shape of the dispersal kernel and the rate and composition of pollen issued from uncharacterized pollen sources. The best dispersal kernel fitted here, the geometric kernel, should now be integrated into models that aim at predicting gene flow at the landscape level, in particular between GM and non-GM crops.     

Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves

Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature.     

甘蓝型油菜pol CMS育性恢复基因对orf224/atp6的转录调控

用 10个线粒体基因探针对波里马细胞质雄性不育 (polimaCMS)三系 1141A(pol) ,1141B(nap)和 1141R(pol)的花蕾线粒体RNA进行了Northern检测。结果表明 ,只有 3个探针atp6、orf2 2 4和orf2 2 2检测到转录本的差异。atp6在可育的 1141B中只转录产生一个丰度很高的 1 1kb转录本 ,在雄性不育的 1141A和pol胞质恢复系 1141R中 ,这个转录本的丰度明显减少并出现了分子量较大的 2个转录本 2 2kb、1 9kb转录本。与 1141A相比 ,恢复系1141R的 2 2kb和 1 9kb转录本丰度明显减少 ,并伴随着两个新的转录本 1 4kb和 1 3kb。表明orf2 2 4 atp6的表达与polCMS有关 ,并且其转录受到恢复基因Rfp的调控。同时通过对杂种F1 ( 1141A× 1141R)与另一个恢复系RS35 (pol)的比较证实 ,Rfp对orf2 2 4 atp6的调控与Rfp纯合与否无关。orf2 2 4 atp6在 1141A的苗期叶片中还转录产生育性恢复特异的 1 4kb转录本 ,这可能与细胞核基因型和相对低温条件有关。