Effect of the epimutagen 5-azacytidine on the structure of floral-stalk metameres in sugar beet (Beta vulgaris L)

This study is dedicated to the effect of the epimutagen 5-azacytidine on the structure of floral-stalk metameres in sugar beet Beta vulgaris L. Simple phytomeres consist of separate flowers (unianthous plants; UA phenotype), while complex metameres occur in synanthous plants (SA phenotype). Treatment of the synanthous line mcSOAN-5 dramatically reduced the number of flowers on phytomeres as early as in generation zero (A0Az0). For the first time, plants with simple phytomeres were found in this line. The proportion of plants with the SA phenotype in generation A0Az0 was 7.7%. In generation A1Az1, the tendency for reduction of the number of flowers on phytomeres persisted. The proportion of plants with simple phytomeres reached 77%; i.e., the frequency of SA phenotypes in the progeny (A1Az1) increased tenfold in comparison with the parents A0Az0. The high frequency of morphogenetic changes in the floral stalk structure under the influence of the epimutagen suggests that the variability of the UA--SA character in beet populations is of epigenetic rather than mutational nature.     

Effect of 5-azacitidine on flower stalk branching in sugar beet (Beta vulgaris L.)

An influence of epimutagen 5-azacitidine on a flower stalk morphogenesis in sugar beet was studied. After the epimutagene treatment the great number of the first- and the third-order branch formation was observed. A higher level of branching completely modified the flower stalk architectonics (generations A0Az0 and A1Az1). A number of the second-order branches in the control and the experimental plants were not distinguished. A new epiphenotype with higher level of branching (generation A0Az0) inherited in daughter generation A1Az1. A flower stalk architectonics was modified because the third-order branches developed in the bract axil instead of flower primordium. A great number of lateral shoot modified a metamer organization of the flower stalk. The metamers on the third-order branches were single-flowered.     

Cytogenetics of autotetraploid sugar beet (Beta vulgaris L.)

Summary Autotetraploid and diploid varieties of sugar beet were investigated for morphology, plant development, root and seed yield. The results obtained from the tetraploid varieties were evaluated according to the number of euploid and aneuploid plants found in each variety. Aneuploid plants often are characterized by delayed growth and poor root or seed yield, which reflects in the average yield of the tetraploid variety. Eutetraploid plants will compete successfully with their diploid counterparts. Until chromosomal stability of euploid plants will be found, aneuploid plants can be eliminated by mechanical selection only, which has to be repeated in each generation. A mechanical selection for euploidy will not only lower the amount of aneuploids in the tetraploid varieties, but also among the triploid hybrid seeds.

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Zusammenfassung Autotetraploide und diploide Zuckerrübensorten wurden auf Morphologie, Wachstum sowie Rüben-und Samenertrag untersucht. Die Ergebnisse der tetraploiden Sorten wurden gemäß den in ihnen gefundenen Anteilen von euploiden und aneuploiden Pflanzen ausgewertet. Aneuploide Pflanzen sind oft durch verlangsamtes Wachstum und schlechten Rüben- oder Samenertrag gekennzeichnet, was sich im Durchschnittsertrag der tetraploiden Sorten widerspiegelt. Eutetraploide Pflanzen können erfolgreich mit den entsprechenden diploiden konkurrieren. Ehe nicht chromosomal stabile eutetraploide Pflanzen gefunden werden, können Aneuploide nur durch mechanische Selektion ausgelesen werden. Diese muß in jeder Generation wiederholt werden. Eine mechanische Selektion auf Aneuploide wird die Anzahl der Aneuploiden nicht nur in den tetraploiden Sorten herabsetzen, sondern auch unter den triploiden Hybridsamen.

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Resumen Se investigó variedades autotetraploides y diploides de remolacha azucarera en relación con la morfología, el desarrollo y el rendimiento en raíz y semilla. Los resultados obtenidos de la variedad tetraploide se evaluaron de acuerdo con el número de plantas euploides y aneuploides halladas. Las plantas aneuploides se caracterizan frecuentemente por un crecimiento retardado y una producción pobre en raíz o semilla, propiedades que se reflejan en el rendimiento medio de las variedades tetraploides. Las plantas eutetraploides pueden competir con éxito con sus correspondientes diploides. Mientras no se alcance la estabilidad cromosómica de las plantas euploides, las aneuploides pueden únicamente eliminarse por medio de una selección mecánica, selección que debe repetirse en cada generación. Una selección mecánica para euploidía no sólo rebajará la proporción de aneuploides en las variedades tetraploides sino tambien dentro de las semillas híbridas triploides.

     

A linkage map of sugar beet (Beta vulgaris L.)

Summary We have established a first linkage map for beets based on RFLP, isozyme and morphological markers. The population studied consisted of 96 F₂ individuals derived from an intraspecific cross. As was expected for outbreeding species, a relatively high degree of polymorphism was found within sugar beet; 47% of the DNA markers were polymorphic for the chosen population. The map consists of 115 independent chromosomal loci designated by 108 genomic DNA probes, 6 isozyme and one morphological marker. The loci cover 789 cM with an average spacing of 6.9 cM. They are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Eighteen markers (15.4%) showed distorted segregation which, in most instances, can be explained by gametic selection of linked lethal loci. The application of the linkage map in sugar beet breeding is discussed.     

Molecular genetic investigation of sugar beet (Beta vulgaris L.)

Molecular genetic studies of sugar beet (Beta vulgaris L.) are reviewed as a basis for the development of genomics of this species. The methods used to study structural and functional genomics are considered. The results and their application to increase the efficiency of sugar beet breeding are discussed.     

Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet)

Summary The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to <5% (<4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20–30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.     

Variability of cathodic peroxidases in sugar beet (Beta vulgaris L.) cultivars

Eighteen varieties of sugar beet (Beta vulgaris L.), originating from various European countries, were compared in respect of peroxidase variability level. They were cultivated in the same experimental plot. The cultivars differed in ploidy level: one variety was tetraploid, three were diploid and 14 varieties were triploid. The cathodic peroxidase system is controlled by four independent genes, of which only one is polymorphic. Consequently, the varieties were characterised by frequencies of 3 allozymes belonging to one locus. Only one variety proved to be fully monomorphic. Genetic similarities between the cultivars were illustrated by a dendrogram (UPGMA) and show different groups of varieties not related to their ploidy level.     

Genetic and chromosomal localization of the 5S rDNA locus in sugar beet (Beta vulgaris L.).

A digoxigenin-labelled 5S rDNA probe containing the 5S rRNA gene and the adjacent intergenic spacer was used for in situ hybridization to metaphase and interphase chromosomes of a trisomic stock from sugar beet (Beta vulgaris L.). Three chromosomes of primary trisomic line IV (T. Butterfass. Z. Bot. 52: 46-77. 1964) revealed signals close to the centromeres. Polymorphisms of 5S rDNA repeats in a segregating population were used to map genetically the 5S rRNA genes within a cluster of markers in linkage group II of sugar beet. The concentration of genetic markers around the centromere presumably reflects the suppressed recombination frequency in centromeric regions. The correlation of physical and genetic data allowed the assignment of a linkage group to sugar beet chromosome IV according to line IV of the primary trisomics.     

The "crystals" in the sugar beet (Beta vulgaris L.) leaves

Crystal containing cells widely distributed in plant tissues, though the origin of the crystals and their functions are still opened to question. Membrane vesicles in beet leaves are visible in electronic microscope. They originate in cytoplasm and penetrate into vacuole by pinocytosis with participation of tonoplast. In light microscope, vesicles are luminous likewise crystals in crystal cells. Such vesicles-"crystals" fulfill crystal cells also. The content of vesicles-"crystals" are electronic transparent at every path of leaf development. It was proposed that distinct vesicles-"crystals" in cytoplasm and vacuole and mass of them in crystal cells, vein bundles, and epidermal cells--all of them are lytic compartments. Later, obviously, true crystals are formed.     

Thidiazuron-induced organogenesis and somatic embryogenesis in sugar beet (Beta vulgaris L.)

Summary Improved in vitro tissue culture systems are needed to facilitate the application of recombinant DNA technology to the improvement of sugar beet germplasm. The effects of N ⁶-benzyladenine (BA) and thidiazuron (TDZ) pretreatment on adventitious shoot and somatic embryogenesis regeneration were evaluated in a range of sugar beet breeding lines and commercial varieties. Petiole explants showed higher frequencies of direct adventitious shoot formation and produced more shoots per explant than leaf lamina explants. TDZ was more effective than BA for the promotion of shoot formation. The optimal TDZ concentrations were 2.3–4.6 μM for the induction of adventitious shoot regeneration. Direct somatic embryogenesis from intact seedlings could be induced by either BA or TDZ. TDZ-induced somatic embryogenesis occurred on the lower surface of cotyledons at concentrations of 0.5–2μM and was less genotype-dependent than with Ba. A high frequency of callus induction could be obtained from seedlings and leaf explants, but only a few of the calluses derived from leaf explants could regenerate to plants via indirect somatic embryogenesis. These results demonstrated that TDZ could prove to be a more effective cytokinin for in vitro culture of sugar beet than BA. Rapid and efficient regeneration of plants using TDZ may provide a route for the production of transgenic sugar beet following Agrobacterium-mediated transformation.     

Biolistic transformation of highly regenerative sugar beet (Beta vulgaris L.) leaves

Leaves of greenhouse-grown sugar beet (Beta vulgaris L.) plants that were first screened for high regeneration potential were transformed via particle bombardment with the uidA gene fused to the osmotin or proteinase inhibitor II gene promoter. Stably transformed calli were recovered as early as 7 weeks after bombardment and GUS-positive shoots regenerated 3 months after bombardment. The efficiency of transformation ranged from 0.9% to 3.7%, and stable integration of the uidA gene into the genome was confirmed by Southern blot analysis. The main advantages of direct bombardment of leaves to regenerate transformed sugar beet include (1) a readily available source of highly regenerative target tissue, (2) minimal tissue culture manipulation before and after bombardment, and (3) the overall rapid regeneration of transgenic shoots.     

Expression of CMS in zygotic and apozygotic progenies of sugar beet Beta vulgaris L

Zygotic and apozygotic progenies of sugar beet exhibit high phenotypic variation with respect to cytoplasmic male sterility (CMS). There are progenies with completely sterile, semisterile, semifertile, and fertile pollen. The proportions of semifertile and fertile plants in zygotic and apozygotic progenies varied from zero to 28% and from zero to 17.8%, respectively. Comparison of the phenotypic distributions in zygotic and apozygotic progenies did not reveal significant differences in the CMS expression, although the latter is determined by the maternal S-plasmotype and both maternal and paternal (pollinator) genotypes in zygotic progenies and only by the maternal S-plasmotype and genotype in apozygotic progenies. It has been hypothesized that the instability of the CMS expression in apozygotic progenies is determined by epigenetic variation in the activities of the genes that control the maintenance of the pollen-grain sterility. Inactivated dominant alleles Rf1(0) and Rf2(0) in homozygous state may function as sterility maintenance genes, whereas activation of these alleles during ontogeny results in a partial or complete restoration of pollen-grain fertility. It was demonstrated that pollen fertility of mother plants with S cytoplasm did not affect the CMS expression in two sib progenies. Conversely, in two other progenies, the proportion of fertile plants was significantly higher in the sib progenies of mother plants with fertile pollen and S cytoplasm (inheritance of epigenetic variation).     

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA

Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.     

Flavin excretion from roots of iron-deficient sugar beet (Beta vulgaris L.)

The characteristics of flavin excretion from iron-deficient sugar-beet roots have been studied. Roots from iron-deficient sugar beet excreted flavins when plants were allowed to decrease the pH of the nutrient solution, but not when plants were grown in nutrient solutions buffered at high pH. As shown by reversed-phase high-performance liquid chromatography, the two major flavins whose excretion was induced by iron deficiency were different from riboflavin, FMN and FAD. These flavins have been identified as riboflavin 3′-sulfate and riboflavin 5′-sulfate by electrospray-mass spectrometry, inductively coupled plasma emission spectroscopy, infrared spectrometry and¹H-nuclear magnetic resonance. We have characterized the time courses of accumulation of the different flavins in the nutrient solution and considered several possible roles for flavin excretion in iron acquisition.     

Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.)

Summary The development of microspore mother cells (MMC) and tapetum in male-fertile and male-sterile anthers of Beta vulgaris L. was compared at the electron microscope level. These studies were complemented by morphometric analyses of mitochondria in both tissues through successive stages of microsporogenesis. The earliest irregularities in the ultrastructure of male-sterile anthers were noted within the tapetum at the tetrad stage. These disturbances were initially expressed by a slight reduction in mitochondrial size and the appearance of concentric configurations of endoplasmic reticulum. As development proceeded, a further decrease in mitochondrial size become more conspicuous and was accompanied by a reduction in ribosome population and a failure of the tapetum to produce Ubisch bodies. This failure to produce Ubisch bodies is reflected in the underdevelopment of sterile microspore exine.     

Genetic analysis of bolting after winter in sugar beet (Beta vulgaris L.)

Key message

This study reveals for the first time a major QTL for post-winter bolting resistance in sugar beet ( Beta vulgaris L.). The knowledge of this QTL is a major contribution towards the development of a winter sugar beet with controlled bolting behavior.

Abstract

In cool temperate climates, sugar beets are currently grown as a spring crop. They are sown in spring and harvested in autumn. Growing sugar beet as a winter crop with an extended vegetation period fails due to bolting after winter. Bolting after winter might be controlled by accumulating genes for post-winter bolting resistance. Previously, we had observed in field experiments a low post-winter bolting rate of 0.5 for sugar beet accession BETA 1773. This accession was crossed with a biennial sugar beet with regular bolting behavior to develop a F₃ mapping population. The population was grown in the greenhouse, exposed to artificial cold treatment for 16 weeks and transplanted to the field. Bolting was recorded twice a week from May until October. Post-winter bolting behavior was assessed by two different factors, bolting delay (determined as days to bolt after cold treatment) and post-winter bolting resistance (bolting rate after winter). For days to bolt, means of F₃ families ranged from 25 to 164 days while for bolting rate F₃ families ranged from 0 to 1. For each factor one QTL explaining about 65 % of the phenotypic variation was mapped to the same region on linkage group 9 with a partially recessive allele increasing bolting delay and post-winter bolting resistance. The results are discussed in relation to the potential use of marker-assisted breeding of winter sugar beets with controlled bolting.     

Effect of cutting on solute uptake by plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves.

The uptake of sucrose, 3-O-methylglucose (3-O-MeG), and valine were studied in discs and in purified plasma membrane vesicles (PMV) prepared from sugar beet (Beta vulgaris L.) exporting leaves. The uptake capacities of freshly excised leaf discs were compared with the uptake in discs that had been floated for 12 h on a simple medium (aging) and with discs excised from leaves that had been cut from the plant 12 h before the experiments (cutting). After cutting, sucrose uptake amounted to twice the uptake measured in fresh discs, whereas the uptake of 3-O-MeG and valine remained unaffected. In aged leaf discs, there was a general stimulation of uptake, which represented 400, 300, and 400% of the uptake measured in fresh discs for sucrose, 3-O-MeG, and valine, respectively. Sucrose uptake in fresh discs was sensitive to N-ethylmaleimide (NEM), to p-chloromercuribenzenesulfonic acid (PCMBS), and to mersalyl acid (MA). Although cutting induced the appearance of a sucrose uptake system that is poorly sensitive to NEM but sensitive to PCMBS and MA, aging induced the development of an uptake system that is sensitive to NEM but poorly sensitive to PCMBS and MA. Autoradiographs of discs fed with [14C]sucrose show that cutting resulted in an increase of vein labeling with little effect in the mesophyll, whereas aging induced an increase of labeling located mainly in the mesophyll. The data show that cutting is sufficient to induce dramatic and selective changes in the uptake properties of leaf tissues and that the effects of cutting and aging on the uptake of organic solutes are clearly different. Parallel experiments were run with purified PMV prepared from fresh and cut leaves. The uptake of sugars and amino acids was studied after imposition of an artificial proton motive force (pmf). Comparison of the uptake properties of PMV and of leaf tissues indicate that the recovery of the sucrose uptake system in PMV is better than the recovery of the hexose and of the valine uptake systems. As observed with the leaf discs, cutting induced a 2-fold increase of the initial rate of sucrose uptake in PMV but did not affect the uptake of valine and 3-O-MeG. Cutting induced an increase of both Vmax and Km of the sucrose transport system in PMV. Measurements of the pmf imposed on the vesicles indicated that the increase of sucrose uptake induced by cutting was not due to a better integrity of the vesicles. Hexoses did not compete with sucrose for uptake in PMV from fresh and cut leaves, and maltose was a stronger inhibitor of sucrose uptake in PMV from cut leaves than in PMV from fresh leaves. The sensitivity of sucrose uptake to NEM, PCMBS, and MA in PMV from fresh and cut leaves paralleled that described above for the corresponding leaf discs. These data show that (a) the changes induced by cutting on sucrose uptake by leaf discs are due to membrane phenomena and not to the metabolism of sucrose; (b) the study of sucrose uptake with PMB gives a good account of the physiological situation; and (c) the specific effects induced by cutting on the sucrose uptake system are not lost during the preparation of the PMV.     

A refined method for ovule culture in sugar beet (Beta vulgaris L.)

Induction of gynogenesis through ovule culture is a valuable tool to produce haploid and doubled haploid plants in sugar beet (Beta vulgaris L.). However, there is still large room for refining the method. In this study we investigated the gynogenic response of cultured ovules of three sugar beet genotypes, the effect of the application to inflorescences of different pretreatments with mannitol at 4ºC and with 5-azacytidine and 2,4-D, and the effect of the use of different basal culture media and sucrose concentrations. The response was evaluated in terms of percentages of induction of gynogenesis, embryogenesis and callogenesis, as well as of regenerated plants. We showed that a pretreatment with 0.5 M mannitol at 4 °C for 4 days, and with 50 µM 5-AzaC for 1 h, notably improved the percentage of embryogenesis and plant regeneration. Besides, the use of MS basal medium and 60 g/L sucrose was also found beneficial. This study provides new ways to improve the efficiency of haploid induction and plant regeneration through ovule culture in sugar beet, and is potentially applicable to ovule culture in other crops.

     

Multiple-line cross quantitative trait locus mapping in sugar beet (Beta vulgaris L.)

Linkage mapping based on multiple-line crosses is a promising strategy for mapping quantitative trait loci (QTL) underlying important agronomic traits. The main goal of this survey was to study the advantages of QTL mapping across versus within biparental populations using experimental data from three connected sugar beet (Beta vulgaris L.) populations evaluated for beet yield and potassium and sodium content. For the combined analysis across populations, we used two approaches for cofactor selection. In Model A, we assumed identical cofactors for every segregating population. In contrast, in Model B we selected cofactors specific for every segregating population. Model A performed better than Model B with respect to the number of QTL detected and the total proportion of phenotypic variance explained. The QTL analyses across populations revealed a substantially higher number of QTL compared to the analyses of single biparental populations. This clearly emphasizes the potential to increase QTL detection power with a joint analysis across biparental populations.     

Effects of zinc toxicity on sugar beet (Beta vulgaris L.) plants grown in hydroponics

The effects of high Zn concentration were investigated in sugar beet ( Beta vulgaris L.) plants grown in a controlled environment in hydroponics. High concentrations of Zn sulphate in the nutrient solution (50, 100 and 300 μ m ) decreased root and shoot fresh and dry mass, and increased root/shoot ratios, when compared to control conditions (1.2 μ m Zn). Plants grown with excess Zn had inward-rolled leaf edges and a damaged and brownish root system, with short lateral roots. High Zn decreased N, Mg, K and Mn concentrations in all plant parts, whereas P and Ca concentrations increased, but only in shoots. Leaves of plants treated with 50 and 100 μ m Zn developed symptoms of Fe deficiency, including decreases in Fe, chlorophyll and carotenoid concentrations, increases in carotenoid/chlorophyll and chlorophyll a / b ratios and de-epoxidation of violaxanthin cycle pigments. Plants grown with 300 μ m Zn had decreased photosystem II efficiency and further growth decreases but did not have leaf Fe deficiency symptoms. Leaf Zn concentrations of plants grown with excess Zn were high but fairly constant (230–260 μg·g⁻¹ dry weight), whereas total Zn uptake per plant decreased markedly with high Zn supply. These data indicate that sugar beet could be a good model to investigate Zn homeostasis mechanisms in plants, but is not an efficient species for Zn phytoremediation.