In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

Aims: To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. Materials and Methods: Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. Results: Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. Conclusions: Arabino-oligosaccharides can be considered as potential prebiotics. Significance and Impact of the Study: Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.

Environmental and human health impacts of growing genetically modified herbicide-tolerant sugar beet: a life-cycle assessment

There is ongoing debate concerning the possible environmental and human health impacts of growing genetically modified (GM) crops. Here, we report the results of a life-cycle assessment (LCA) comparing the environmental and human health impacts of conventional sugar beet growing regimes in the UK and Germany with those that might be expected if GM herbicide-tolerant (to glyphosate) sugar beet is commercialized. The results presented for a number of environmental and human health impact categories suggest that growing the GM herbicide-tolerant crop would be less harmful to the environment and human health than growing the conventional crop, largely due to lower emissions from herbicide manufacture, transport and field operations. Emissions contributing to negative environmental impacts, such as global warming, ozone depletion, ecotoxicity of water and acidification and nutrification of soil and water, were much lower for the herbicide-tolerant crop than for the conventional crop. Emissions contributing to summer smog, toxic particulate matter and carcinogenicity, which have negative human health impacts, were also substantially lower for the herbicide-tolerant crop. The environmental and human health impacts of growing GM crops need to be assessed on a case-by-case basis using a holistic approach. LCA is a valuable technique for helping to undertake such assessments.

An application of life-cycle assessment for environmental planning and management: the potential environmental and human health impacts of growing genetically-modified herbicide-tolerant sugar beet

Life-Cycle Assessment (LCA) was used to assess the potential environmental and human health impacts of growing genetically-modified (GM), herbicide-tolerant sugar beet in the UK and Germany compared with conventional sugar beet varieties. The GM variety results in lower potential environmental impacts on global warming, airborne nutrification, ecotoxicity (of soil and water) and watercourse enrichment, and lower potential human health impacts in terms of production of toxic particulates, summer smog, carcinogens and ozone depletion. Although the overall contribution of GM sugar beet to reducing harmful emissions to the environment would be relatively small, the potential for GM crops to reduce pollution from agriculture, including diffuse water pollution, is highlighted.

The effect of wheat straw residue on the emergence and early growth of sugar beet (Beta vulgaris) and oilseed rape (Brassica napus)

The management of straw residue can be a concern in non-inversion tillage systems where straw tends to be incorporated at shallow depths or left on the soil surface. This can lead to poor crop establishment because straw residue can impede or hinder crop emergence and growth. Small container-based experiments were undertaken using varying amounts of wheat straw residue either incorporated or placed oil the soil surface. The effects on (lays to seedling emergence, percentage emergence, seedling dry-weight and soil temperature using sugar beet and oilseed rape were investigated because these crops often follow wheat in a cropping sequence. The position of the straw residue was found to be the primary factor in reducing crop emergence and growth. Increasing the amount of straw residue (from 3.3 t ha(-1) to 6.7 t ha(-1)) did not show any consistent trends in reducing crop emergence or growth. However, in some instances, results indicated that an interaction between the position and the amount of straw residue Occurred particularly when the straw and seed was placed on the soil surface. Straw placed on the soil surface significantly reduced mean day-time soil temperature by approximately 2.5 degrees C compared to no residue. When the seed and straw was placed on the soil Surface a lack of seed-to-soil contact caused a reduction in emergence by approximately 30% because of the restriction in available moisture that limited the ability for seed imbibition. This trend was reversed when the seed was placed in the soil, but with straw residue still on the soil surface, because the surface straw was likely to reduce moisture evaporation and improved seed-to-soil contact that led to rapid emergence. In general, when straw was mixed in or placed on the soil surface along with the seed, sugar beet and oilseed rape emergence and early growth biomass was significantly restricted by approximately 50% compared to no residue. The consequences of placing seed with or near to straw residue have been shown to cause a restriction in crop establishment. In both oilseed tape and sugar beet, this could lead to a reduction in final crop densities, poor, uneven growth and potentially lower yields that could lower financial margins. Therefore, if farmers are planning to use non-inversion tillage methods for crop establishment, the management and removal of straw residue from near or above the seed is considered important for successful crop establishment. (C) 2008 Elsevier B.V. All rights reserved.

Soil disturbed using a strip tillage implement on a range of soil types and the effects on sugar beet establishment

The area of soil disturbed using a single tine is well documented. However, modern strip tillage implements using a tine and disc design have not been assessed in the UK or in mainland Europe. Using a strip tillage implement has potential benefits for European agriculture where economic returns and sustainability are key issues. Using a strip tillage system a narrow zone is cultivated leaving most of the straw residue on the soil surface. Small field plot experiments were undertaken on three soil types and the operating parameters of forward speed, tine depth and tine design were investigated together with measurements of seedbed tilth and crop emergence. The type of tine used was found to be the primary factor in achieving the required volume of disturbance within a narrow zone whilst maintaining an area of undisturbed soil with straw residue on the surface. The winged tine produced greater disturbance at a given depth compared with the knife tine. Increasing forward speed did not consistently increase the volume of disturbance. In a sandy clay loam the tilth created and emergence of sugar beet by strip tillage and ploughing were similar but on a sandy loam the strip tillage treatments generally gave a finer tilth but poorer emergence particularly at greater working depth.

Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics

Background The rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere. Results Carbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere. Conclusions Comparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants.

Towards more precise sugar beet management based on geostatistical analysis of spatial variability within fields

Within-field variation in sugar beet yield and quality was investigated in three commercial sugar beet fields in the east of England to identify the main associated variables and to examine the possibility of predicting yield early in the season with a view to spatially variable management of sugar beet crops. Irregular grid sampling with some purposively-located nested samples was applied. It revealed the spatial variability in each sugar beet field efficiently. In geostatistical analyses, most variograms were isotropic with moderate to strong spatial dependency indicating a significant spatial variation in sugar beet yield and associated growth and environmental variables in all directions within each field. The Kriged maps showed spatial patterns of yield variability within each field and visual association with the maps of other variables. This was confirmed by redundancy analyses and Pearson correlation coefficients. The main variables associated with yield variability were soil type, organic matter, soil moisture, weed density and canopy temperature. Kriged maps of final yield variability were strongly related to that in crop canopy cover, LAI and intercepted solar radiation early in the growing season, and the yield maps of previous crops. Therefore, yield maps of previous crops together with early assessment of sugar beet growth may make an early prediction of within-field variability in sugar beet yield possible. The Broom’s Barn sugar beet model failed to account for the spatial variability in sugar yield, but the simulation was greatly improved when corrected for early canopy development cover and when the simulated yield was adjusted for weeds and plant population. Further research to optimize inputs to maximise sugar yield should target the irrigation and fertilizing of areas within fields with low canopy cover early in the season.

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Stomatal guard cells are totipotent

It has been successfully demonstrated, using epidermis explants of sugar beet (Beta vulgaris L.), that stomatal guard cells retain full totipotent capacity. Despite having one of the highest degrees of morphological adaptation and a unique physiological specialization, it is possible to induce a re-expression of full (embryogenic) genetic potential in these cells in situ by reversing their highly differentiated nature to produce regenerated plants via a callus stage. The importance of these findings both to stomatal research and to our understanding of cytodifferentiation in plants is discussed.

Quantitative response of the longevity of seed of twelve crops to temperature and moisture in hermetic storage

Seeds of 39 seed lots of a total of twelve different crops were stored hermetically in a wide range of air-dry environments (2-25% moisture content at 0-50 degrees C), viability assessed periodically, and the seed viability equation constants estimated. Within a species, estimates of the constants which quantify absolute longevity (K-E) and the relative effects on longevity of moisture content (C-W) and temperature (C-H and C-Q) did not differ (P >0.05 to P >0.25) among lots. Comparison among the 12 crops provided variant estimates of K-E and C-W (P< 0.01), but common values of C-H and C-Q (0.0322 and 0.000454, respectively, P >0.25). Maize (Zea mays) provided the greatest estimate of K-E (9.993, s.e.= 0.456), followed by sorghum (Sorghum bicolor) (9.381, s.e. 0.428), pearl millet (Pennisetum typhoides) (9.336, s.e.= 0.408), sugar beet (Beta vulgaris) (8.988, s.e.= 0.387), African rice (Oryza glaberrima) (8.786, s.e.= 0.484), wheat (Triticum aestivum) (8.498, s.e.= 0.431), foxtail millet (Setaria italica) (8.478, s.e.= 0.396), sugarcane (Saccharum sp.) (8.454, s.e.= 0.545), finger millet (Eleusine coracana) (8.288, s.e.= 0.392), kodo millet (Paspalum scrobiculatum) (8.138, s.e.= 0.418), rice (Oryza sativa) (8.096, s.e.= 0.416) and potato (Solanum tuberosum) (8.037, s.e.= 0.397). Similarly, estimates of C-W were ranked maize (5.993, s.e.= 0.392), pearl millet (5.540, s.e.= 0.348), sorghum (5.379, s.e.=0.365), potato (5.152, s.e.= 0.347), sugar beet (4.969, s.e.= 0.328), sugar cane (4.964, s.e.= 0.518), foxtail millet (4.829, s.e.= 0.339), wheat (4.836, s.e.= 0.366), African rice (4.727, s.e.= 0.416), kodo millet (4.435, s.e.= 0.360), finger millet (4.345, s.e.= 0.336) and rice (4.246, s.e.= 0.355). The application of these constants to long-term seed storage is discussed.

PGR forum: serving the crop wild relative user community

The Euro-Mediterranean region is an important centre for the diversity of crop wild relatives. Crops, such as oats (Avena sativa), sugar beet (Beta vulgaris), apple (Malus domestica), annual meadow grass (Festuca pratensis), white clover (Trifolium repens), arnica (Arnica montana), asparagus (Asparagus officinalis), lettuce (Lactuca sativa), and sage (Salvia officinalis) etc., all have wild relatives in the region. The European Community funded project, PGR Forum (www.pgrforum.org) is building an online information system to provide access to crop wild relative data to a broad user community; including plant breeders, protected area managers, policy-makers, conservationists, taxonomists and the wider public. The system will include data on uses, geographical distribution, biology, population and habitat information, threats (including IUCN Red List assessments) and conservation actions. This information is vital for the continued sustainable utilisation and conservation of crop wild relatives. Two major databases have been utilised as the backbone to a Euro-Mediterranean crop wild relative catalogue, which forms the core of the information system: Euro+Med PlantBase (www.euromed.org.uk) and Mansfeld’s World Database of Agricultural and Horticultural Crops (http://mansfeld.ipk-gatersleben.de). By matching the genera found within the two databases, a preliminary list of crop wild relatives has been produced. Around 20,000 of the 30,000+ species listed in Euro+Med PlantBase can be considered crop wild relatives, i.e. those species found within the same genus as a crop. The list is currently being refined by implementing a priority ranking system based on the degree of relatedness of taxa to the associated crop.

Vertical distribution, size and composition of the weed seedbank under various tillage and herbicide treatments in a sequence of industrial crops

Investigations were conducted during the 2003, 2004 and 2005 growing seasons in northern Greece to evaluate effects of tillage regime (mouldboard plough, chisel plough and rotary tiller), cropping sequence (continuous cotton, cotton-sugar beet rotation and continuous tobacco) and herbicide treatment on weed seedbank dynamics. Amaranthus spp. and Portulaca oleracea were the most abundant species, ranging from 76% to 89% of total weed seeds found in 0-15 and 15-30 cm soil depths during the 3 years. With the mouldboard plough, 48% and 52% of the weed seedbank was found in the 0-15 and 15-30 cm soil horizons, while approximately 60% was concentrated in the upper 15 cm soil horizon for chisel plough and rotary tillage. Mouldboard ploughing significantly buried more Echinochloa crus-galli seeds in the 15-30 cm soil horizon compared with the other tillage regimes. Total seedbank (0-30 cm) of P. oleracea was significantly reduced in cotton-sugar beet rotation compared with cotton and tobacco monocultures, while the opposite occurred for E. crus-galli. Total seed densities of most annual broad-leaved weed species (Amaranthus spp., P. oleracea, Solanum nigrum) and E. crus-galli were lower in herbicide treated than in untreated plots. The results suggest that in light textured soils, conventional tillage with herbicide use gradually reduces seed density of small seeded weed species in the top 15 cm over several years. In contrast, crop rotation with the early established sugar beet favours spring-germinating grass weed species, but also prevents establishment of summer-germinating weed species by the early developing crop canopy.

Method of plant tissue culture and regeneration

Plants may be regenerated from stomatal cells or protoplasts of such cells. Prior to regeneration the cells or protoplasts may be genetically transformed by the introduction of hereditary material most preferably by a DNA construct which is free of genes which specify resistance to antibiotics. The regeneration step may include callus formation on a hormone-free medium. The method is particularly suitable for sugar beet.

Technologies for biological containment of GM and Non-GM crops

International Perspective The development of GM technology continues to expand into increasing numbers of crops and conferred traits. Inevitably, the focus remains on the major field crops of soybean, maize, cotton, oilseed rape and potato with introduced genes conferring herbicide tolerance and/or pest resistance. Although there are comparatively few GM crops that have been commercialised to date, GM versions of 172 plant species have been grown in field trials in 31 countries. European Crops with Containment Issues Of the 20 main crops in the EU there are four for which GM varieties are commercially available (cotton, maize for animal feed and forage, and oilseed rape). Fourteen have GM varieties in field trials (bread wheat, barley, durum wheat, sunflower, oats, potatoes, sugar beet, grapes, alfalfa, olives, field peas, clover, apples, rice) and two have GM varieties still in development (rye, triticale). Many of these crops have hybridisation potential with wild and weedy relatives in the European flora (bread wheat, barley, oilseed rape, durum wheat, oats, sugar beet and grapes), with escapes (sunflower); and all have potential to cross-pollinate fields non-GM crops. Several fodder crops, forestry trees, grasses and ornamentals have varieties in field trials and these too may hybridise with wild relatives in the European flora (alfalfa, clover, lupin, silver birch, sweet chestnut, Norway spruce, Scots pine, poplar, elm, Agrostis canina, A. stolonifera, Festuca arundinacea, Lolium perenne, L. multiflorum, statice and rose). All these crops will require containment strategies to be in place if it is deemed necessary to prevent transgene movement to wild relatives and non-GM crops. Current Containment Strategies A wide variety of GM containment strategies are currently under development, with a particular focus on crops expressing pharmaceutical products. Physical containment in greenhouses and growth rooms is suitable for some crops (tomatoes, lettuce) and for research purposes. Aquatic bioreactors of some non-crop species (algae, moss, and duckweed) expressing pharmaceutical products have been adopted by some biotechnology companies. There are obvious limitations of the scale of physical containment strategies, addressed in part by the development of large underground facilities in the US and Canada. The additional resources required to grow plants underground incurs high costs that in the long term may negate any advantage of GM for commercial productioNatural genetic containment has been adopted by some companies through the selection of either non-food/feed crops (algae, moss, duckweed) as bio-pharming platforms or organisms with no wild relatives present in the local flora (safflower in the Americas). The expression of pharmaceutical products in leafy crops (tobacco, alfalfa, lettuce, spinach) enables growth and harvesting prior to and in the absence of flowering. Transgenically controlled containment strategies range in their approach and degree of development. Plastid transformation is relatively well developed but is not suited to all traits or crops and does not offer complete containment. Male sterility is well developed across a range of plants but has limitations in its application for fruit/seed bearing crops. It has been adopted in some commercial lines of oilseed rape despite not preventing escape via seed. Conditional lethality can be used to prevent flowering or seed development following the application of a chemical inducer, but requires 100% induction of the trait and sufficient application of the inducer to all plants. Equally, inducible expression of the GM trait requires equally stringent application conditions. Such a method will contain the trait but will allow the escape of a non-functioning transgene. Seed lethality (‘terminator’ technology) is the only strategy at present that prevents transgene movement via seed, but due to public opinion against the concept it has never been trialled in the field and is no longer under commercial development. Methods to control flowering and fruit development such as apomixis and cleistogamy will prevent crop-to-wild and wild-to-crop pollination, but in nature both of these strategies are complex and leaky. None of the genes controlling these traits have as yet been identified or characterised and therefore have not been transgenically introduced into crop species. Neither of these strategies will prevent transgene escape via seed and any feral apomicts that form are arguably more likely to become invasives. Transgene mitigation reduces the fitness of initial hybrids and so prevents stable introgression of transgenes into wild populations. However, it does not prevent initial formation of hybrids or spread to non-GM crops. Such strategies could be detrimental to wild populations and have not yet been demonstrated in the field. Similarly, auxotrophy prevents persistence of escapes and hybrids containing the transgene in an uncontrolled environment, but does not prevent transgene movement from the crop. Recoverable block of function, intein trans-splicing and transgene excision all use recombinases to modify the transgene in planta either to induce expression or to prevent it. All require optimal conditions and 100% accuracy to function and none have been tested under field conditions as yet. All will contain the GM trait but all will allow some non-native DNA to escape to wild populations or to non-GM crops. There are particular issues with GM trees and grasses as both are largely undomesticated, wind pollinated and perennial, thus providing many opportunities for hybridisation. Some species of both trees and grass are also capable of vegetative propagation without sexual reproduction. There are additional concerns regarding the weedy nature of many grass species and the long-term stability of GM traits across the life span of trees. Transgene stability and conferred sterility are difficult to trial in trees as most field trials are only conducted during the juvenile phase of tree growth. Bio-pharming of pharmaceutical and industrial compounds in plants Bio-pharming of pharmaceutical and industrial compounds in plants offers an attractive alternative to mammalian-based pharmaceutical and vaccine production. Several plantbased products are already on the market (Prodigene’s avidin, β-glucuronidase, trypsin generated in GM maize; Ventria’s lactoferrin generated in GM rice). Numerous products are in clinical trials (collagen, antibodies against tooth decay and non-Hodgkin’s lymphoma from tobacco; human gastric lipase, therapeutic enzymes, dietary supplements from maize; Hepatitis B and Norwalk virus vaccines from potato; rabies vaccines from spinach; dietary supplements from Arabidopsis). The initial production platforms for plant-based pharmaceuticals were selected from conventional crops, largely because an established knowledge base already existed. Tobacco and other leafy crops such as alfalfa, lettuce and spinach are widely used as leaves can be harvested and no flowering is required. Many of these crops can be grown in contained greenhouses. Potato is also widely used and can also be grown in contained conditions. The introduction of morphological markers may aid in the recognition and traceability of crops expressing pharmaceutical products. Plant cells or plant parts may be transformed and maintained in culture to produce recombinant products in a contained environment. Plant cells in suspension or in vitro, roots, root cells and guttation fluid from leaves may be engineered to secrete proteins that may be harvested in a continuous, non-destructive manner. Most strategies in this category remain developmental and have not been commercially adopted at present. Transient expression produces GM compounds from non-GM plants via the utilisation of bacterial or viral vectors. These vectors introduce the trait into specific tissues of whole plants or plant parts, but do not insert them into the heritable genome. There are some limitations of scale and the field release of such crops will require the regulation of the vector. However, several companies have several transiently expressed products in clinical and pre-clinical trials from crops raised in physical containment.