In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Studies on allometric growth relationships of sugar beet(Beta vulgaris) grown in nutrient culture

The effects of genotype and seed size on the growth of sugar beetseedlings in liquid nutrient culture medium were examined. Seed size waspositively correlated to seedling weight at the 5/7-leaf stage of developmentbut had little influence on mean total root to shoot ratio. At the 8-leaf-stagethere was considerable variation in the fibrous root to shoot ratios betweenindividuals in each of three cultivars. Nevertheless, the mean root to shootratio of seedlings of each cultivar was relatively similar. However, largegenotypic differences were detected in the depletion of specific elements fromthe nutrient culture solution, this being particularly so for nitrogen,potassium and sodium.     

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.     

Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.     

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.     

Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

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.     

Genetic localization of four genes for nematode (Heterodera schachtii Schm.) resistance in sugar beet (Beta vulgaris L.)

Sugar beet (Beta vulgaris L.) is highly susceptible to the beet cyst nematode (Heterodera schachtii Schm.). Three resistance genes originating from the wild beets B. procumbens (Hs1 ^pro-1) and B. webbiana (Hs1 ^web-1, Hs2 ^web-7) have been transferred to sugar beet via species hybridization. We describe the genetic localization of the nematode resistance genes in four different sugar beet lines using segregating F₂ populations and RFLP markers from our current sugar beet linkage map. The mapping studies yielded a surprising result. Although the four parental lines carrying the wild beet translocations were not related to each other, the four genes mapped to the same locus in sugar beet independent of the original translocation event. Close linkage (0–4.6 cM) was found with marker loci at one end of linkage group IV. In two populations, RFLP loci showed segregation distortion due to gametic selection. For the first time, the non-randomness of the translocation process promoting gene transfer from the wild beet to the sugar beet is demonstrated. The data suggest that the resistance genes were incorporated into the sugar beet chromosomes by non-allelic homologous recombination. The finding that the different resistance genes are allelic will have major implications on future attempts to breed sugar beet combining the different resistance genes.     

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.     

Influence of long-term drought stress on osmolyte accumulation in sugar beet (Beta vulgaris L.) plants

Accumulation of various osmolytes was examined in plants of sugar beet cv. Janus grown under two soil water treatments: control (60% of the field water capacity; FWC) and drought (30–35% FWC). The water shortage started on the 61st day after emergence (DAE), at the stage of the beginning of tap-roots development and was imposed for 35 days. Osmotic potential of sugar beet plant organs, particularly tap-roots, was decreased significantly as a consequence of a long-term drought. Water shortage reduced univalent (K⁺, Na⁺) cations concentrations in the petioles and divalent (Ca²⁺, Mg²⁺) ions level in the mature and old leaves. Cation concentrations in the tap-roots were not affected by water shortage. The ratio of univalent to divalent cations was significantly increased in young leaves and petioles as a consequence of drought. Long-term water deficit caused a significant reduction of inorganic phosphorus (P_i) concentration in young and old leaves. Under the water stress condition, the concentration of proline was increased in all individual plant organs, except proline concentration in the youngest leaves. Drought treatment caused a significant increase of glycine betaine content in shoot without any change in tap-roots. Glucose concentrations were significantly increased only in tap-roots as the effect of drought. In response to water shortage the accumulation of sucrose was observed in all the examined leaves and tap-roots. Overall, a long-term drought activated an effective mechanism for osmotic adjustment both in the shoot and in the root tissues which may be critical to survival rather than to maintain plant growth but sugar beet organs accumulate different solutes as a response to water cessation.     

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.)

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes” and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

Short-term effects of Al3+ on the osmotic behavior of red beet (Beta vulgaris L.) protoplasts

The short-term (less than 10 min) effects of Al³⁺ on the biophysical properties of plasma membranes were investigated by time-series image analysis of osmotically-induced volumetric and morphologic changes of red beet (Beta vulgaris L.) protoplasts. Exposure to Al³⁺ under hypotonic conditions reduced the volumetric expansion of protoplasts and their resultant burst: i.e. lysis of protoplasts in a concentration-dependent manner. Under hypertonic conditions, protoplasts exposed to Al³⁺ underwent an enhanced volumetric contraction in cross-sectional area, while maintaining higher protoplast roundness. The residual effects of Al³⁺ pre-treatment on subsequent osmotic behavior were also examined, and protoplasts pre-treated with Al³⁺ also exhibited less lysis during subsequent exposure to hypotonic conditions and enhanced volumetric contractions and higher roundness under subsequent hypertonic conditions. Under our experimental conditions, Al³⁺ consistently minimized protoplast surface area by inhibiting osmotic expansion or by enhancing osmotic contraction, as well as by maintaining higher protoplast roundness. These results suggested that the electrostatic property of Al³⁺ might have induced the binding and possible cross-linking of negatively-charged sites on the plasma membrane surface. This may be an important factor in understanding the mechanism of Al³⁺ phytotoxicity.     

Genetical control and linkage relationships of isozyme markers in sugar beet (B. vulgaris L.)

Summary Five isozyme systems were genetically investigated. The different separation techniques, the developmental expression and the use as marker system in sugar beet genetics and breeding is discussed. Isocitrate dehydrogenase was controlled by two genes. The gene products form inter- as well as intralocus dimers, even with the gene products of the Icd gene in B. procumbens and B. patellaris. Adenylate kinase was controlled by one gene. Three different allelic forms were detected, which were active as monomeric proteins. Glucose phosphate isomerase showed two zones of activity. One zone was polymorphic. Three allelic variants, active as dimers, were found. Phosphoglucomutase also showed two major zones of activity. One zone was polymorphic and coded for monomeric enzymes. Two allelic forms were found in the accessions studied. The cathodal peroxidase system was controlled by two independent genes, of which only one was polymorphic. The gene products are active as monomers. Linkage was found between red hypocotyl color (R) and Icd ₂. Pgm ₁, Gpi ₂, Ak ₁ and the Icd ₂-R linkage group segregated independently.     

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998     

Monosomic additions in beet (Beta vulgaris) carrying extra chromosomes of Beta procumbens

Summary Eleven isozyme systems were used to identify the extra chromosomes, originating from Beta procumbens, in progenies of 33 monosomic additions in beet (B. vulgaris). Nine groups of monosomic additions could be distinguished, representing the nine different chromosome types of B. procumbens.     

Phytohormone changes during storage root growth in Beta species

The physiological and morphological factors necessary for efficient accumulation of sucrose in sugar beet (Beta vulgaris L.) are considered in relation to potential uses of plant growth regulators to modify the anatomy of storage roots so as to increase sucrose content and yield. The percentage of sucrose in root fresh and dry matter is closely related to root structure. Sugar beet, mangold and chard are three sub-species of Beta vulgaris that differ considerably in their anatomy, assimilate partitioning, sucrose concentration and root dry matter yield. The concentrations of indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins were measured during the growth of the storage root in each of these cultivars. Correlations were found between the phytohormone levels and the formation of secondary cambia and their subsequent cell division and expansion activity.     

Intraspecific diversity of sugar beet (Beta vulgaris) mitochondrial DNA

Summary Mitochondrial (mt) DNA, isolated from different sugar beet populations, was analyzed using BamHI and EcoRI restriction enzymes. It was shown that plants possessing the new mtDNA types are revealed among O-type fertilizers quite frequently. Among cytoplasmic male sterile (cms) plants, which evolved during cultivation of O-type fertilizers, plants with altered mt genome were found.     

dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.