Ecological aspects of transgenic sugar beet: transfer and expression of herbicide resistance in hybrids with wild beets

Summary An increasing number of genetically engineered cultivars of several crops is being experimentally released into the environment. In future, crops with new transgenic traits will probably play an important role in agricultural practice. The long-term effect of transgenes on community ecology will depend on the distribution and establishment of transgenic plants in the wild, on the sexual transfer of their new genes to the environment and on the potential ecological impact of the transgenic trait. The starting point was the use of transgenic sugar beet lines, Beta vulgaris subspec. vulgaris var. altissima DÖLL (Helm 1957), with transgenes coding for rhizomania and herbicide (BASTA^®) resistance. The first two questions to answer were: Can the transgenes be transferred via pollen to wild beets, Beta vulgaris subspec. maritima (L.) ARCANG. or cultivated relatives such as red beet or spinach beet and are they expressed in the hybrids? Can transgenes be monitored in young Beta vulgaris-hybrids? The experimental transfer of transgenes was conducted in 1993 at a field location in northern Germany. The beets were hand-pollinated with transgenic pollen. In a non destructive biotest, the hybrid seedlings were tested for herbicide resistance. Transgenic plants showed no noxious phenotypic effects whereas control plants developed leaf necroses. All herbicide resistant hybrids within the biotest were assumed to be transgenic.     

QTL for delayed bolting after winter detected in leaf beet (Beta vulgaris L.)

Growing sugar beet (Beta vulgaris L. ssp. vulgaris) as a winter crop in cool temperate climates is expected to increase yield potential. However, this requires bolting resistance after winter. One strategy to achieve complete bolting resistance is to accumulate genes for bolting delay from various genetic resources within the B. vulgaris gene pool. To identify such genes, a QTL mapping was performed in a segregating population derived from a biennial leaf beet with delayed bolting after winter. The population was tested for bolting delay after winter in two different experiments with natural or artificial vernalization. Three QTL for bolting delay were mapped on linkage groups 3, 5 and 9 affecting bolting time by up to 19 days. These QTL could be combined with recently reported bolting QTL to develop a winter sugar beet with complete bolting resistance.     

The Diploidised Meiosis of Tetraploid Beta macrocarpa and its Possible Application in Breeding Sugar Beet

Accessions of Beta macrocarpa Guss., collected on the Canary Islands, were tetraploid (2n = 36). Three of these accessions were studied in detail. The plants were rather uniform in their morphological appearance, both within and between accessions, and were annual and fully self-compatible. Meiosis was completely diploidised, suggesting an alloploid nature of the tetraploid species. Crosses with diploid B. vulgaris yielded triploid hybrids which were sterile or nearly so; a few descendants of such hybrids were highly aneuploid. Crosses between tetraploid B. macrocarpa and autotetraploid cytotypes of B. vulgaris showed variable results, only part of these crosses yielded tetraploid hybrids. The tetraploid hybrids exhibited somewhat higher fertility than the triploids. An F₂ generation showed partial hybrid dwarfness, partial fertility and segregation for carliness and coloration of the hypocotyl. All hybrids had multivalents at meiosis (averages: 2.5—5.4 multivalents per pollen mother cell), indicating suppression of the diploidised meiosis. The possibilities of application of the diploidised meiosis in breeding sugar beet are discussed.     

Crosses between Beta intermedia bunge and B. vulgaris L.

Summary A tetraploid annual male sterile form of Beta vulgaris L. (2n=4x=36) was crossed with the wild beet species Beta intermedia Bunge (2n=36). The resulting F₁-plants were male sterile annuals being two or three times back-crossed to diploid and tetraploid sugar and fodder beets in the next years. Apart from tetraploid material (36 chromosomes) hexaploid (54 chromosomes) and a number of aneuploid plants developed.The results obtained justify the conclusion that, at a tetraploid level the material mostly propagates apomictically after the F₁ generation. The presence of penta-, hexa-, septa-and even octaploid plants might be explained by assuming that no meiosis has taken place in the crossing partners. Triploid plants are sometimes found in the progeny of hexaploid material and may presumably be considered haploids. Moreover some pentaploid plants were found in the progeny of the open pollinated F₁ which after two generations of bagging are still pentaploids although they produce no pollen. This is another clear indication of apomictic reproduction.The tetraploid generation from the cross between the hexaploid material and diploid sugar beets probably contains the best prospects for breeding.     

Evaluation of genetic diversity and root traits of sea beet accessions of the Adriatic Sea coast

Thirty-nine sea beet [Beta vulgaris L. ssp. maritima (L.) Arcang.] accessions of the Adriatic coast were screened genetically and for their adaptive morpho-functional root traits in order to identify new sources of abiotic resistances for sugar beet breeding programs. Genetic diversity was evaluated with 21 microsatellites markers that identified 44 polymorphic alleles. Sea beets grouped into two main clusters: the West and the East Adriatic coast groups, with the latter showing higher genetic diversity. Among sea beet accessions with desirable root traits, four accessions have proved to be interesting for sugar beet [B. vulgaris (L.) ssp. vulgaris] breeding aimed to improve tolerance to nutritional stresses. Lastovo (ID 29) and Zut (ID 34) accessions were characterized by the highest values of RER, TRL, FRL and RSA still maintaining a high value of RTD, while Grado (ID 21) an Portic (ID 23) accessions were characterized by the highest RTD, but with low values of RER, TRL, FRL and RSA parameters.     

Variation in some agronomically important characters in a germplasm collection of beet (Beta vulgaris L.)

Summary A geographically representative selection of germplasm of Beta vulgaris, section Beta has been assessed for characteristics important in sugarbeet breeding, including downy mildew resistance, resistance to aphid colonisation and infection by the beet virus yellow complex. The occurrence of maintainer lines for cytoplasmic male-steriles was also investigated. Desirable qualities were found in some accessions, including nothern European wild vulgaris ssp. maritima and some old multigerm cultivars of fodder beets.     

Crosses between Beta vulgaris L. and Beta lomatogona F. et M.

Summary A population of 2x^ms sugar beets was crossed with 4x Beta lomatogona F. et M. The 3x F₁-plants were male sterile and were backcrossed with 2x and 4x sugar beets and multiplied without pollination as well. After the 1st backcross mainly 3x apomict types arose again and, among others, a small number of successful 4x backcrosses. After pollination by 4x sugar beets this 4x F₁ B₁ produced. besides predominatly apomictically multiplied 4x plants, also about 7% haploid 2x hybrids. The latter probably possess 1 genome from B. vulgaris and 1 genome from B. lomatogona. In the meiosis of the PMC's a certain amount of homeology between a number of chromosomes of both species could be established. The amphihaploid hybrids can be used as breeding parents for the creation of types in which introgession can occur. During hybridization in addition to 2x and 4x B. vulgaris types a number of 2x-, 3x-, 5x- and 6x-hybrids arose. This is presumably caused by the presence of gametes with the somatic number of chromosomes and the occurrence sometimes of haploid apomictic multiplication.The presence of large numbers of bolters in the F₁ and F₁ B₁ suggests that the bolting tendency of both species is based on different genes.     

Peroxidase activity as a possible test for distinguishing s-plasm from n-plasm in sugar beet (Beta vulgaris L.)

Summary The O-type lines (N-plasm) of sugar beet (Beta vulgaris L.) proved to have a consistently lower peroxidase activity (49.2–62.7%) than their male sterile (MS) equivalents (S-plasm).Similar differences in peroxidase activity (45.4–56.3%) were found when O-type lines were compared with unrelated MS lines. They were also observed in different parts of the plant (cotyledons, hypocotyl + radicle, true leaves) and were reproducible.The fertile lines could be grouped by their level of peroxidase activity in categories of either S-plasm or N-plasm.The peroxidase activity test could be used for selection of N-plasm lines from fertile sugar beet populations and for selection of MS and O-type lines of higher stability.     

A genetic map of sugar beet (Beta vulgaris) based on RAPD markers

Linkage analysis of sugar beet (Beta vulgaris L.) was performed with random amplified polymorphic DNA (RAPD)-markers. From three segregating populations, a combined genetic map was constructed which comprises 85 RAPD, five isozyme, one RFLP marker and the genes for resistance against the nematode Heterodera schachtii Schm., one restorer locus for male sterility and the genes for annuality and hypocotyl colour. For mapping of the two unlinked restorer genes a statistical model was developed based on the maximum-likelihood function.     

Beet mild yellowing virus resistance derived from wild and cultivated Beta germplasm

Sugar beet (Beta vulgaris ssp. vulgaris L.) has a relatively narrow genetic base and several programmes have sought to evaluate the potential for introducing novel traits from wild and cultivated Beta germplasm into the crop. In particular, resistance to important sugar beet diseases has been identified within individual Beta accessions. We report here the successful transfer of resistance to Beet mild yellowing virus (BMYV) from garden beet, fodder beet and leaf beet accessions to progeny populations in initial crosses with sugar beet. Twelve plant populations derived from different Beta accessions were inoculated with viruliferous aphids carrying BMYV and the virus content of individual plants subsequently quantified by an ELISA test. Seven populations were significantly more resistant than a control sugar beet cultivar (P ≤ 0.05). BMYV resistance was successfully inherited in BC₁ and BC₂ generations, suggesting that resistance could potentially be introgressed from these sources into elite sugar beet lines.     

Use of haploids of Beta vulgaris L. for the study of orcein and giemsa stained chromosomes

Summary Some haploids isolated among the progeny from crosses involving several genotypically different motherplants of Beta vulgaris L., were used for an investigation on chromosome morphology of the genus Beta.The length of both the long and short arms proved to vary considerably for each chromosome, so that a representative idiogram of the 9 chromosomes could not be made. Staining the chromosomes according to the BSG-technique revealed only bands in the centromere regions. However, one chromosome with a narrow band in the short arm could be identified, which was supposed to be the nucleolar chromosome. The C-bands presumably correlate with the heterochromatic blocks as observed in mitotic and meiotic (pachytene) prophase.This study was part of a project on cytogenetics of interspecific hybrids resistent to beet cyst eelworm, supported by the Institute of Rational Sugar Production,Bergen op Zoom, the Netherlands.     

Gene flow in common bean (Phaseolus vulgaris L.)

This study was conducted in Brazil in order to assess the potential risk posed by gene escape from transgenic into non-transgenic plants and wild populations. A new methodology was applied to evaluate the gene flow between common bean cultivars, by means of a specially delineated experiment in two stages. The first stage consisted of the planting of one cultivar with violet flowers (BB) as pollen source (‘Diamante Negro’), and a receiver (‘Talismã’) with white flowers (bb), at different distances. The source was sown in the center of the area. The pollen receiver cultivar was sown, in concentrical squares around it. At maturity, the rows were sampled at varied distances from the source in the four cardinal directions. In the second stage, the sampled seeds of the previous stage were sown, and the percentage of outcrossing was evaluated during flowering through the presence of violet flowers (Bb). The highest frequency of natural hybrids, 0.136%, occurred at a distance of 0.5 m between the cultivars. The natural outcrossing rate was practically zero beyond a distance of 3.25 m.     

Molecular and morpho-physiological characterization of sea,ruderal and cultivated beets

Beta vulgaris genetic resources are essential for broadening genetic base of sugar beet and developing cultivars adapted to adverse environmental conditions. Wild beets (sea beets, B. vulgaris spp. maritima and their naturalized introgressions with cultivated beets known as ruderal beets) harbor substantial genetic diversity that could be useful for beet improvement. Here, we compared molecular and morpho-physiological traits of wild beets collected on the Adriatic coast of Italy with sugar beet using eight primer-pairs amplifying 194 polymorphic fragments and four root traits (glucose and fructose content in the root tip, root elongation rate, number of the of root tips, total root length and its distribution among diameters ranges). Genetic diversity was higher in the sea beet accession, which may be due to the highly variable selection pressures that occur in heterogeneous ecological niches, compared with the ruderal and cultivated beets. Sea and sugar beet accessions showed contrasting root patterns in response to sulfate deprivation: sugar beet showed an increase of reducing sugars in the root tips and higher root elongation rate, and the sea beet accession showed an increase in root tip number, total root length and fine root length (average diameter < 0.5 mm). The ruderal beet showed intermediary responses to sea and sugar beet accessions. AFLP and morpho-physiological cluster analyzes showed sea, ruderal and cultivated beets to be genetically distinct groups. The results of this study indicate variability in response to sulfate deprivation is present in undomesticated beets that could be deployed for sugar beet improvement.     

Sugar beets homozygous for resistance to beet cyst nematode (Heterodera schachtii Schm.), developed from monosomic additions of Beta procumbens to B. vulgaris

Summary Monosomic additions of Beta vulgaris x B. procumbens with resistance to beet cyst nematode (Heterodera schachtii Schm.) were used for the production of resistant diploids through incorporation of the B. procumbens chromosome fragment bearing the resistance gene(s) into one of the sugar beet chromosomes. The heterozygotes obtained accordingly were selfed for producing homozygotes. These homozygotes differed morphologically from commercial sugar beet varieties, but produced reasonable amounts of pollen. Female transmission of resistance was 100%, whereas male transmission, apart from some exceptions, was more than 90%. The number of hypersensitivity reactions to penetrated larvae was related to the degree of susceptibility. Larval development was severely retarded in the resistant plants, preventing most of them to produce cysts. If cysts were formed, their content was considerably less as compared to those in the susceptible plants.     

Out-crossing frequency and genetic analysis of hybrids between transgenic glufosinate herbicide-resistant rice and the weed, red rice

The potential of transferring herbicide resistance from transgenic rice (Oryza sativa L.) varieties to sexually compatible weeds is of paramount importance for development of effective weed control strategies. The objective of this research was to determine the genetic control and frequency of natural outcrossing between a transgenic, glufosinate-resistant rice line and a Louisiana biotype of red rice (Oryza sativa L.). Molecular and phenotypic data showed that outcrossing in field plots between a non-transgenic purple marker line and red rice did occur within one field season, but at a low rate of <1%. Similarly, molecular and phenotypic data demonstrated that glufosinate resistance was transferred from the transgenic line to the red rice biotype in the field within one year at a low frequency of 0.3%. Compared to parental lines, the transgenic-red rice hybrids were extremely late, tall, and never set seed during the normal field season. Genetic analyses in all F₂ populations showed glufosinate resistance behaved in a Mendelian fashion as a single, dominant gene. Presence of the bar gene for glufosinate resistance did not increase fitness or seed fecundity in hybrids or subsequent progeny. The genetic analyses and outcrossing results from this study suggest that an effective management program can be developed to prolong the usefulness of transgenic, glufosinate herbicide technology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Determination of the Ploidy Level of Pure and Mixed Plant Populations of Sugar Beet (Beta vulgaris L.) by Flow Cytometry

To improve estimates of the true ploidy level of pure diploid triploid, and tetraploid sugar beet (Beta vulgaris L.) plants, and of populations of mixed ploidy, the effect of leaf age/size was evaluated on ploidy determination by flow cytometry. There were significant differences between old and young leaves, with young leaves giving the best estimate of the true ploidy level of the plants. The old leaves seemed to express a high degree of endopolyploidy.     

Production of Haploid Sugar Beet (Beta vulgaris L.) by Culturing Unpollinated Ovules

The culture of unpollinated ovules is shown to be a suitable system for the production of haploid sugar beet (Beta vulgaris L.). The yield of haploids depended upon the genotype and varied between 0 and 13 % with a mean of 1.0 %. Haploid plants could be produced from approximately 50 % of all genotypes examined. The majority of the haploids isolated (about 90%) maintained the haploid genome level during the in vitro culture and propagation; 10% of the haploid clones showed a spontaneous doubling to the diploid genome level.     

A marker-assisted analysis of bolting tendency in sugar beet (Beta vulgaris L.)

Bolting tendency in sugar beet varies among breeding lines and cultivars. Four crosses were made between breeding lines susceptible and resistant to bolting in order to study the genetic basis of bolting tendency. Bolting percentage in F₂, after 8 weeks of low temperature treatment, varied among the crosses, suggesting a complicated genetic control of bolting tendency. Different segregation ratios were observed, in particular, between families derived from the bolting F₁ plants and those from their non-bolting siblings, the former families showing a significantly higher bolting percentage than the latter. A marker-assisted analysis with seven isozyme loci, Ak1, Gdh2, Idh1, Lap, Mdh1, Pgi2 and Pgm1, revealed that a locus with marked effect on bolting tendency was located near Idh1. Because of a close linkage of Idh1 with B for annuality, the gene tagged by Idh1 appeared to be equivalent or similar to B′ for easy bolting allelic to B as reported by Owen et al. (1940). The results obtained suggest that the B locus may not only have an important role in determining growth habits but also control various degrees of bolting tendency in individual sugar beet plants. A linked pair, Ak1-Lap, and Pgi2 also were found to affect bolting tendency, although their effects varied depending on the crosses and families tested. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular markers linked to rhizomania resistance in sugar beet, Beta vulgaris, from two different sources map to the same linkage group

Rhizomania, one of the most important diseases of sugar beet, is caused by beet necrotic yellow vein virus, a Furovirus vectored by the fungus Polymyxa betae Keskin. Reduction of the production losses caused by this disease can only be achieved by using tolerant cultivars. The objective of this study was the identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to a rhizomania resistance gene. The RAPD markers were identified using bulked segregant analysis in a segregating population of 62 individuals derived by intercrossing plants of the resistant commercial hybrid GOLF, and the resistance locus was positioned in a molecular marker linkage map made with a different population of 50 GOLF plants. The resistance locus, Rr1, was mapped to linkage group III of our map of Beta vulgaris L. ssp. vulgaris, which consisted of 76 RAPDs, 20 restriction fragment length polymorphisms (RFLPs), three sequence characterized amplified regions (SCARs) and one sequence tagged site (STS). In total, 101 molecular markers were mapped over 14 linkage groups which spanned 688.4 cM with an average interval length of 8.0 cM. In the combined map, Rr1 proved to be flanked by the RAPD loci RA411₁₈₀₀ and AS7₁₁₀₀ at 9.5 and 18.5cM, respectively. Moreover, in our I₂ population, we found that a set of markers shown by Barzen et al. (1997) to be linked to the ‘Holly’ type resistance gene was also linked to the ‘GOLF’-type resistance gene. These results appeared to indicate that the rhizomania resistance gene present in the GOLF hybrid could be the same gene underlying resistance in ‘Holly’-based resistant genotypes. Two other explanations could be applied: first, that two different alleles at the same locus could have been selected; second, that two different genes at two different but clustered loci underwent the selection process.