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.     

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.     

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.     

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.     

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.     

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.     

Identification of a sucrose nonfermenting-1-related protein kinase in sugar beet (Beta vulgaris L.)

A 154 bp polymerase chain reaction product, SBKIN154, showing 76–83% sequence identity with sucrose nonfermenting-1 (SNF1)-related protein kinase nucleotide sequences from other plant species was amplified from sugar beet storage root RNA. Southern blot analysis using SBKIN154 as a hybridisation probe suggested that sugar beet contains either a single-copy SNF1-related gene or a small gene family of highly conserved genes. An antibody raised to a heterologously-expressed fusion of the rye SNF1-related protein kinase, RKIN1, and maltose binding protein, recognised a protein of the expected size (M_r approx. 60,000) on western blots of storage root, stalk, leaf and root extracts. Measurements of SNF1-related activity were made using a specific peptide (SAMS) phosphorylation assay. Activity was highest (0.38 nmol min^-1 mg^-1 protein) in developing storage roots and lowest (0.035 nmol min^-1 mg^-1) in fibrous roots.     

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.     

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.     

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.     

Evaluation of nematode-resistant sugar beet (Beta vulgaris L.) lines by molecular analysis

 Thirty sugar beet (Beta vulgaris) lines conferring complete resistance to the beet cyst nematode (BCN, Heterodera schachtii) originating from interspecific crosses with wild beets of the section Procumbentes (B. procumbens, B. webbiana and B. patellaris) were investigated by morphology and wild beet-specific molecular markers. The beet lines carrying chromosome mutations consisted of monosomic additions (2n=18+1), fragment additions (2n=18+fragment) and translocations (2n=18) from the wild beets. Genome-specific single-copy, satellite and repetitive probes were applied to study the origin, chromosomal assignment and presence of nematode resistance genes. Within the wild beet species at least three different resistance genes located on different chromosomes were distinguished: Hs1 on the homoelogous chromosomes I of each species, Hs2 on the homoelogous chromosomes VII of B. procumbens and B. webbiana and Hs3 on chromosome VIII of B. webbiana. A clear distinction between the three chromosomes was possible by morphological and molecular means. The translocation lines were separated into two different groups: one containing the resistance gene Hs1 from chromosome I and the other carrying a different nematode resistance gene. The molecular data combined with sequence analyses of Hs1 of the three wild beet species revealed a clear distinction between B. procumbens and B. webbiana. The evolutionary and taxonomical relationship of these species supporting the idea of three different species originating from a common ancestor is discussed. Received: 6 April 1998 / Accepted: 22 April 1998     

Morpho-physiological responses of sugar beet (Beta vulgaris L.) genotypes to drought stress

The identification of morpho-physiological traits related to drought tolerance and high yield potential is a challenge when selecting sugar beet genotypes with greater tolerance to water stress. In this paper, root morphological parameters, antioxidant systems, leaf relative water content (RWC) and H⁺-ATPase activity as key morpho-physiological traits involved in drought tolerance/susceptibility of sugar beet were studied. Genotypes showing a different drought tolerance index (DTI) but a similar yield potential, under moderate (?0.6 Mpa) and severe (?1.2 MPa) water stress, were selected and their morpho-physiological traits were investigated. The results showed a wide genetic variation in morpho-physiological parameters which demonstrated the different adaptive strategies under moderate and severe drought conditions in sugar beet. In particular, an efficient antioxidant system and redox signalling made some sugar beet genotypes more tolerant to drought stress. The alternative strategy of other genotypes was the reduction of root tissue density, which produced a less dense root system improving the axial hydraulic conductivity. These results could be considered as interesting challenge for a better understanding of the drought tolerance mechanisms in sugar beet.     

Survey of sugar beet (Beta vulgaris L.) hAT transposons and MITE-like hATpin derivatives

Genome-wide analyses of repetitive DNA suggest a significant impact particularly of transposable elements on genome size and evolution of virtually all eukaryotic organisms. In this study, we analyzed the abundance and diversity of the hAT transposon superfamily of the sugar beet (B. vulgaris) genome, using molecular, bioinformatic and cytogenetic approaches. We identified 81 transposase-coding sequences, three of which are part of structurally intact but nonfunctional hAT transposons (BvhAT), in a B. vulgaris BAC library as well as in whole genome sequencing-derived data sets. Additionally, 116 complete and 497 truncated non-autonomous BvhAT derivatives lacking the transposase gene were in silico-detected. The 116 complete derivatives were subdivided into four BvhATpin groups each characterized by a distinct terminal inverted repeat motif. Both BvhAT and BvhATpin transposons are specific for species of the genus Beta and closely related species, showing a localization on B. vulgaris chromosomes predominantely in euchromatic regions. The lack of any BvhAT transposase function together with the high degree of degeneration observed for the BvhAT and the BvhATpin genomic fraction contrasts with the abundance and activity of autonomous and non-autonomous hAT transposons revealed in other plant species. This indicates a possible genus-specific structural and functional repression of the hAT transposon superfamily during Beta diversification and evolution.     

Responses of sugar beet (Beta vulgaris L.) to drought and nutrient deficiency stress

The responses of two sugar beet genotypes, 24367 (putative droughttolerant) and N6 (putative drought intolerant), to drought and nutrientdeficiency stress were investigated in an attempt to identify reliable andsensitive indicators of stress tolerance. In glasshouse-grown plants of bothgenotypes, relative water content (RWC) of the leaves decreased and leaftemperature increased in response to drought stress. Genotype differences inresponse to drought included leaf RWC, glycine betaine accumulation, alterationof shoot/root ratio and production of fibrous roots. Thus, in comparison to N6,genotype 24367 lost less water from leaves, produced more fibrous roots,produced more glycine betaine in shoots and tap roots and had a much reducedshoot/root ratio in response to withholding water for up to 215 h.The hydraulic conductance and sap flow of sugar beet seedlings grown innutrientculture decreased when subjected to nitrogen deficiency stress. Under nitrogensufficient conditions sap flow was greater in 24367 than in N6. The resultsindicate that genotype 24367 is more tolerant to stresses induced by water andnitrogen deficiency and that increased fibrous root development may be a majorfactor in increasing sap flow via a concomitant enhancement of aquaporinactivity.     

Expression of Mdh1 locus alleles in apozygotic progenies of sugar beet (Beta vulgaris L.)

Expression of Mdh1 alleles has been studied in 60 apozygotic (agamospermic) sugar beet progenies. Seed progenies were obtained by uniparental (pollen less) mode of seed reproduction: selfing of pollen-sterile plants isolated with paper bags. The apozygotic seed progenies demonstrate a disomic gamete autosegregation, i.e., the ratio between genotypes in the progenies correspond to the gamete segregation in a duplex heterozygote of an autotetraploid. It was shown that the ratio between the Mdh1 phenotypes in apozygotic progenies is strongly affected by spontaneous inactivation of one of the alleles. In most progenies, the excess of FF phenotypes and the deficit of SS phenotypes were observed. In our opinion, such deviations in genotype and phenotype frequencies result from conversion of the active Mdh1-S into the inactive Mdh1-S0 allele (epigenetic gene inactivation). The spontaneous inactivation of one allele results in extremely variable frequencies of heterozygous Mdh1-F/Mdh1-S genotypes and phenotypes in the apozygotic seed progenies. The empirical distribution of the frequencies of heterozygous genotypes in the apozygotic seed progenies is given by a negative binomial distribution describing the expected time of occurrence of random events.     

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.