Construction of a sugar beet BAC library from a hybrid with diverse traits

A bacterial artificial chromosome (BAC) library of the 750-Mbp sugar beet genome represented in hybrid US H20 was constructed fromHind III-digested DNA, with an average insert size of 120 kbp. US H20 is a variety grown in the eastern United States. It exhibits heterosis for emergence and yield, presumably because of its hybridity between eastern and western US germplasm sources. Filter arrays were used to assess the abundance and distribution of particular nucleotide sequences. An rRNA gene probe found that 1.2% of the library carried sequences similar to these highly repetitive and conserved sequences. A simple sequence repeat element (CA)₈ thought to be predominantly distributed throughout centromere regions of all chromosomes was present in 1.7% of clones. For more than half of the 28 randomly chosen expressed sequence tags (ESTs) used as probes, a higher-than-expected number of single-copy hybridization signals was observed. Assuming 6× genome coverage, this suggests that many duplicate genes exist in the beet genome.     

Competitiveness of transgenic sugar beet resistant to beet necrotic yellow vein virus and potential impact on wild beet populations

Beets are a crop of particular concern regarding invasiveness questions because they commonly become feral due to unintentional hybridization with annual forms of wild beets. In this study the performance of transgenic beets resistant to Beet Necrotic Yellow Vein Virus (BNYVV) was compared to the performance of unmodified material from the same breeding line. Both transgenic and control genotypes were also compared to a conventionally bred variety carrying a similar phenotypic trait. Field tests were developed in a step by step fashion in order to study seed emergence and competitiveness in early life stages. The tests quantified the potential ecological advantage of virus resistance under virus and non-virus infestation conditions. In experimental field releases in 1993 and 1994 in Germany, a small but increasingly clear 'additive' ecological advantage of the genetically engineered trait was detected. In both years and all competition treatments, the conventional tolerant variety performed best. An impact of naturalization on natural, nonagricultural habitats may appear in wild beet populations in Italian seed beet production areas. However, a survey of coastal areas of North-Eastern Italy found no virus infestation in 1994, suggesting that an increase in wild beet fitness is unlikely to occur.     

Analysis of root images from auger sampling with a fast procedure: a case of application to sugar beet

Manual line-intersect methods for estimating root length are being progressively replaced by faster and more accurate image analysis procedures. These methods even allow the estimation of some more root parameters (e.g., diameter), but still require preliminary labour-intensive operations. Through a task-specific macro function written in a general-purpose image analysis programme (KS 300 – Zeiss), the processing time of root images was greatly reduced with respect to skeletonisation methods by using a high-precision algorithm (Fibrelength). This has been previously proposed by other authors, and estimates length as a function of perimeter and area of the digital image of roots. One-bit binary images were acquired, aiming at large savings in computer memory, and automatic discrimination of roots against extraneous objects based on their elongation index (perimeter²/area), was performed successfully. Of four tested spatial resolutions (2.9, 5.9, 8.8, 11.8 pixel mm^–1), in clean samples good accuracy in root length estimation was achieved at 11.8 pixel mm^–1, up to a root density of 5 cm cm^–2 on the scanner bed. This resolution is theoretically suitable for representing roots at least 85 m wide. When dealing with uncleaned samples, a thick layer of water was useful in speeding up spreading of roots on the scanner bed and avoiding underestimation of their length due to overlaps with organic debris. A set of fibrous root samples of sugar beet (Beta vulgaris var. saccharifera L.) collected at harvest over two years at Legnaro (NE Italy) was analysed by applying the above procedure. Fertilisation with 100 kg ha^–1 of nitrogen led to higher RLD (root length density in soil) in shallow layers with respect to unfertilised controls, whereas thicker roots were found deeper than 80 cm of soil without nitrogen.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).