Genetic evidence for the origin of Californian wild beets (genus Beta)

The genus Beta L. is a morphologically and genetically variable group composed of wild, weedy, and domesticated forms that are used for sugar production or as vegetables. In this study, we have evaluated genetic variation in 64 germplasm accessions of wild and domesticated beets and examined the origin of wild beet accessions in California using allozyme analysis. UPGMA analysis showed overall that domesticated and wild beets form genetically coherent groups. Wild beets in California have two different origins, from European Beta vulgaris or from Beta macrocarpa. Population-level patterns of allozyme variation for wild California beets related to B. vulgaris suggest that those populations evolved from naturalized populations of the cultivated B. vulgaris ssp. vulgaris which had hybridized to varying degrees with the sea beets B. vulgaris ssp. maritima. Wild California beets related to B. macrocarpa are essentially genetically identical to European accessions. In addition, we found substantial evidence for hybridization and introgression of B. vulgaris alleles in one B. macrocarpa accession in California. The obligate outcrosser B. vulgaris exhibits more allelic diversity than the self-compatible B. macrocarpa. Beta vulgaris ssp. maritima exhibits more genetic diversity than domesticated B. vulgaris ssp. vulgaris. Received: 2 November 1998 / Accepted: 29 April 1999     

Polymorphic microsatellite markers for inferring diversity in wild and domesticated sugar beet (Beta vulgaris)

Eight microsatellite loci were characterized within two cultivated beet (Beta vulgaris ssp. vulgaris) accessions and one accession of the wild progenitor of domesticated sugar beet, Beta vulgaris ssp. maritima. Allele diversity was high, yielding two to 11 alleles per locus. Polymorphism information content (PIC) values obtained for these eight loci where also high and indicate the highly informative nature of the microsatellites presented here. These described markers add to a small set of publicly available microsatellite markers for beet and will be instrumental in identifying patterns of genetic diversity and origins of domestication.     

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     

Influence of sugar beet breeding on populations of Beta vulgaris ssp. maritima in Italy

Abstract. It is highly probable that transgenic cultivars of sugar beet may influence wild beets in the seed-production-area of northern Italy. For this reason a survey of the local wild beet populations and their habitat characteristics was conducted in 1994/1995, i.e. before transgenic beets and their off spring could have become established. Wild beets (Beta vulgaris ssp. maritima) were found at 21 locations between Trieste and Cesenatico, as part of the natural littoral vegetation classified as Atriplicetum tatarici (Cakiletea maritimae) and Crithmetum (Crithmo-Staticetea). The analysis of phenotypic attributes leads to a division into three different sub-populations. Greenhouse studies on the morphology and life-cycle attributes demonstrated actual gene flow between conventional seed beet and the examined wild beet population.     

Nuclear and cytoplasmic genetic diversity in weed beet and sugar beet accessions compared to wild relatives: new insights into the genetic relationships within the <Emphasis Type="Italic">Beta vulgaris</Emphasis> complex species

Hybridization between cultivated species and their wild relatives is now widely considered to be common. In the Beta vulgaris complex, the sugar beet seed multiplication areas have been the scene of inadvertent pollination of sugar beet seed bearers by wild ruderal pollen donors, generating a weedy form of beet which infests sugar beet fields in European countries. Up to now, investigations of evolutionary dynamics of genetic diversity within the B. vulgaris complex were addressed using few genetical markers and few accessions. In this study, we tackled this issue using a panel of complementary markers: five nuclear microsatellite loci, four mitochondrial minisatellite loci and one chloroplastic PCR-RFLP marker. We sampled 1,640 individuals that illustrate the actual distribution of inland ruderal beets of South Western France, weed beets and wild sea beets of northern France as well as the diversity of 35 contemporary European diploid cultivars. Nuclear genetic diversity in weed beets appeared to be as high as those of ruderal beets and sea beets, whereas the narrowness of cultivar accessions was confirmed. This genetic bottleneck in cultivars is even more important in the cytoplasmic genome as only one haplotype was found among all sugar beet cultivars. The large majority of weed beet populations also presented this unique cytoplasmic haplotype, as expected owing to their maternal cultivated origin. Nonetheless, various cytoplasmic haplotypes were found within three populations of weed beets, implying wild-to-weed seed flows. Finally, our findings gave new insights into the genetical relationships between the components of the B. vulgaris complex: (1) we found a very strong genetic divergence between wild sea beet and other relatives, which was unexpected given the recent evolutionary history and the full cross-compatibility of all taxa and (2) we definitely confirmed that the classification into cultivated, wild, ruderal and weed forms according to their geographical location, phenotype or their domesticated status is clearly in accordance with genetic clustering despite the very recent domestication process of sugar beet. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic diversity and gene flow between wild, cultivated and weedy forms of Beta vulgaris L. (Chenopodiaceae), assessed by RFLP and microsatellite markers

 Beets belonging to the species Beta vulgaris L. can be found in crop, wild and weedy forms, all of which are interfertile. We studied the intra-specific genetic relationships of about 300 individuals from 54 populations of various French geographic origins using nuclear molecular markers (five single-copy RFLP loci and one microsatellite locus). The patterns of diversity were congruent for both types of markers. Genetic diversity in wild beets appeared to be high, both in term of allele number and observed heterozygosity, whereas the narrowness of the cultivated-beet gene pool was confirmed. Genetic distances between all forms showed that weed beets in northern France are intermediates between sugar beet and inland wild beets in south-western France. This analysis allowed us to infer the paternal origin of weed beets and furthermore, is in agreement with a previous study which focused on their maternal origin: weed beet infesting sugar-beet fields originated from accidental and recurrent hybridization between cultivated lines and ruderal inland wild beets during the production of commercial seeds in south-western France. Inland wild beets are genetically close to Mediterranean coastal wild beets, but differ from other coastal forms (from Biscay, Brittany and northern France). The study of gene flow in the beet complex contributes to the risk assessment of transgenic beets. Received: 8 June 1998 / Accepted: 8 October 1998     

Development of simple sequence repeat (SSR) markers for the assessment of gene flow between sea beet (Beta vulgaris ssp. maritima) populations

Molecular markers can be used to estimate gene flow indirectly by monitoring the relative frequency of alleles in adjacent populations. Sea beet (Beta vulgaris ssp. maritima) is a wild plant species found along the coastlines of many European countries and is closely related to cultivated beets. A set of six simple sequence repeat (SSR) markers that are polymorphic in UK populations have been developed for sea beet to assess the problems of indirect measurement of gene flow in these populations.     

Impact of gene flow from cultivated beet on genetic diversity of wild sea beet populations

Gene flow and introgression from cultivated plants may have important consequences for the conservation of wild plant populations. Cultivated beets (sugar beet, red beet and Swiss chard: Beta vulgaris ssp. vulgaris) are of particular concern because they are cross-compatible with the wild taxon, sea beet (B.vs. ssp. maritima). Cultivated beet seed production areas are sometimes adjacent to sea beet populations; the numbers of flowering individuals in the former typically outnumber those in the populations of the latter. In such situations, gene flow from cultivated beets has the potential to alter the genetic composition of the nearby wild populations. In this study we measured isozyme allele frequencies of 11 polymorphic loci in 26 accessions of cultivated beet, in 20 sea beet accessions growing near a cultivated beet seed production region in northeastern Italy, and 19 wild beet accessions growing far from seed production areas. We found one allele that is specific to sugar beet, relative to other cultivated types, and a second that has a much higher frequency in Swiss chard and red beet than in sugar beet. Both alleles are typically rare in sea beet populations that are distant from seed production areas, but both are common in those that are near the Italian cultivated beet seed production region, supporting the contention that gene flow from the crop to the wild species can be substantial when both grow in proximity. Interestingly, the introgressed populations have higher genetic diversity than those that are isolated from the crop. The crop-to-wild gene flow rates are unknown, as are the fitness consequences of such alleles in the wild. Thus, we are unable to assess the long-term impact of such introgression. However, it is clear that gene flow from a crop to a wild taxon does not necessarily result in a decrease in the genetic diversity of the native plant.     

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.     

A Family of Differentially Amplified Repetitive DNA Sequences in the Genus Beta Reveals Genetic Variation in Beta vulgaris Subspecies and Cultivars

Members of a highly abundant restriction satellite family have been isolated from the wild beet species Beta nana. The satellite DNA sequence is characterized by a conserved RsaI restriction site and is present in three of four sections of the genus Beta, namely Nanae, Corollinae, and Beta. It was not detected in species of the evolutionary old section Procumbentes, suggesting its amplification after separation of this section. Sequences of eight monomers were aligned revealing a size variation from 209 to 233 bp and an AT content ranging from 56.5% to 60.5%. The similarity between monomers in B. nana varied from 77.7% to 92.2%. Diverged subfamilies were identified by sequence analysis and Southern hybridization. A comparative study of this repetitive DNA element by fluorescent in situ hybridization and Southern analyses in three representative species was performed showing a variable genomic organization and heterogeneous localizations along metaphase chromosomes both within and between species. In B. nana the copy number of this satellite, with some 30,000 per haploid genome, is more than tenfold higher than in Beta lomatogona and up to 200 times higher than in Beta vulgaris, indicating different levels of sequence amplification during evolution in the genus Beta. In sugar beet (B. vulgaris), the large-scale organization of this tandem repeat was examined by pulsed-field gel electrophoresis. Southern hybridization to genomic DNA digested with DraI demonstrated that satellite arrays are located in AT-rich regions and the tandem repeat is a useful probe for the detection of genetic variation in closely related B. vulgaris cultivars, accessions, and subspecies. Received: 24 May 1996 / Accepted: 13 September 1996     

DNA methylation of retrotransposons,DNA transposons and genes in sugar beet (Beta vulgaris L.)

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome‐wide cytosine methylation in the sugar beet genome was studied in leaves and leaf‐derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome‐wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.     

Phenotypic polymorphism and allele differentiation of isozymes in fodder beet,multigerm sugar beet and monogerm sugar beet

Summary Thirteen enzymes (MDH, SDH, LAP, PGM, PX, IDH, GPI, 6PGD, APH, GOT, GDH, ME and SOD) of 3 cultivated beet (B. vulgaris L.) gene pools, comprising 12 accessions of fodder beet, 11 of old multigerm sugar beet and 10 of modern monogerm sugar beet, were investigated using horizontal starch gel electrophoresis. Eleven accessions of primitive or wild B. vulgaris were also included for the comparison of isozymes. Variation in isozyme phenotypes was investigated to detect diversity in the three cultivated forms of beet. Phenotypic variation was observed in all except ME and SOD, which were monomorphic. A high degree of phenotypic polymorphism (Pj) was found in GDH, PGM, IDH, APH and MDH. Differences in phenotypic polymorphism in MDH, GPI and PX were recognized between fodder beet and both sugar beet groups. Average polymorphism for 13 enzymes in both sugar beets was significantly higher than that in fodder beet. For 13 enzymes, the existence of high isozyme diversity in both sugar beet gene pools was revealed. Allele frequencies in 13 alleles of five enzyme-coding loci, Lap, Px-1, Aph-1, Got-2 and Gdh-2, were investigated. New alleles, Px-1 ¹ and Got-2 ¹, were found in fodder beet accessions. No significant differences of average allele frequencies of five loci between fodder beet and both sugar beets were recognized. Several unique alleles and different isozyme phenotypes were observed in the accessions of B. vulgaris ssp. macrocarpa and ssp. adanensis. Future utilization of cultivated beet gene pools for sugar beet breeding is discussed from the viewpoint of genetic resources.     

Crop-weed interactions in the Beta vulgaris complex at a local scale: allelic diversity and gene flow within sugar beet fields

Crop-wild hybrids and weed beets are the main source of agronomic concern for sugar beet production all over Europe. In order to understand the dynamics of crop-wild interactions and the evolution of weediness in Beta vulgaris, we investigated genetic features of bolting individuals occurring at a local scale, i.e. within two sugar beet fields of the French northern area of sugar beet production. By analysing ploidy level, mitochondrial DNA and microsatellite polymorphism, the genetic diversity and the genetic relationships among three different classes of individuals (variety, in-row and out-row weed-beets) from a given field were examined. Such genetic analyses provide a unique opportunity to obtain evidence for the weeds origin and the evolutionary hypotheses previously stated. All the individuals shared in common the Svulg mitochondrial haplotype, and thus a common maternal origin. Conversely, the large genetic diversity at microsatellite loci highlighted the large diversity of the pollinator plants (cultivated and wild plants) during the-seed production process, as well as during the further evolution of weed beets in the sugar production area. Received: 23 April 2001 / Accepted: 15 June 2001     

A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome‐arm identification,integration of genetic linkage groups and analysis of major repeat family distribution

We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North–South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome‐specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S‐5.8S‐25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber‐FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

High-resolution mapping of YACs and the single-copy gene Hs1pro−1 on Beta vulgaris chromosomes by multi-colour fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful approach for physical mapping of DNA sequences along plant chromosomes. Nematode-resistant sugar beets (Beta vulgaris) carrying aBeta procumbens translocation were investigated by FISH with two differentially labelled YACs originating from the translocation. At mitotic metaphases, the translocation was identified with both YACs in the terminal region on a pair of chromosomes. Meiotic chromosomes, representing a far more extended hybridization target, were used to determine the orientation of YACs with respect to chromosomal domains in combination with chromosomal landmark probes for telomeres and centromeres. The in situ detection of plant single-copy sequences is technically difficult, and the wild beet translocation was used to explore the potential resolution of the FISH approach and to introduce the chromosomal mapping of single-copy genes into genome analysis of Beta species. An internal fragment of the nematode resistance gene Hs1 ^pro–1, 684 bp long, was detected on both chromatids of different Beta chromosomes and represents one of the shortest unique DNA sequences localized on mitotic plant chromosomes so far. Comparative chromosomal mapping of the 684 bp Hs1 ^pro–1 probe in the translocation line, a monosomic addition line and in B. procumbens revealed the origin of the wild beet translocation leading to nematode-resistant sugar beets.     

Nucleotide‐binding resistance gene signatures in sugar beet,insights from a new reference genome

Nucleotide‐binding (NB‐ARC), leucine‐rich‐repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long‐read sequencing, are improving resolution of repeat‐rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB‐ARC domain as a model, 231 tentative NB‐ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB‐ARC signatures as well as full‐length sequences. The NB‐ARC‐associated proteins contained NLR resistance gene domains, including TIR, CC and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB‐ARC domains to validated R‐genes from monocots and eudicots suggested extensive Beta vulgaris‐specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR‐like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic‐acid‐based method for NLR prediction in non‐model plant species.     

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root‐knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest‐resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.     

Chromosomal assignment of the nine linkage groups of sugar beet (Beta vulgaris L.) using primary trisomics

 Twenty-four marker loci representing each of the nine linkage groups of sugar beet (Beta vulgaris) have been assigned to the nine primary trisomics of Butterfass (1964). Single-copy RFLP probes were hybridized with filter-bound DNA of the trisomics. The autoradiographs were scanned and analyzed by densitometric methods. Statistics on the integrated optical densities of the RFLP bands revealed a clear relationship of each linkage group to a distinct trisomic type. For the first time each of the linkage groups could unequivocally be assigned to one sugar beet chromosome. A standard nomenclature of the 9 chromosomes of sugar beet is suggested and discussed with respect to previous numbering systems. Received: 27 February 1997/Accepted: 7 March 1997     

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