Expression in vitro of resistance to Heterodera schachtii in hairy roots of an alien monotelosomic addition plant of Beta vulgaris,transformed by Agrobacterium rhizogenes

Summary Hairy roots, induced by Agrobacterium rhizogenes, were obtained of nematode susceptible beet plants (Beta vulgaris) and of the nematode resistant alien monotelosomic addition AN5, carrying a telosome from B. patellaris. The additional telosome was found to be stably present in vitro in the roots of AN5. The hairy root cultures were inoculated with larvae of the beet cyst nematode Heterodera schachtii. On the root culture of AN5 significantly less cysts developed than on the other root cultures. These results indicate that the resistance to the beet cyst nematode is expressed in the roots after transformation and can be monitored under in vitro conditions.     

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.     

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.     

Breeding for resistance to rhizomania in sugar beet: A review

Currently rhizomania is the most important disease in sugar beet worldwide, and attack can lead to serious yield losses. The disease is caused by beet necrotic yellow vein virus (BNYVV) that is transmitted by the soil-borne fungus Polymyxa betae. Breeding sugar beet cultivars with resistance to rhizomania is regarded as the most appropriate way to enable continued production of this crop in BNYVV-infested fields and also to slow the spread of the disease. Breeding for resistance started with selection by scoring disease symptoms in field experiments. The development of non-destructive greenhouse tests, with determination of the virus concentration in rootlets using ELISA, has greatly improved the efficiency of selection. In this paper the impact of scientific research on the progress in breeding cultivars with resistance to rhizomania is reviewed. This includes the distribution, composition, and pathogenicity of the virus, the sources of resistance to virus and vector, the genetics of virus resistance, progress with breeding methods, and the use of molecular markers and pathogen-derived resistance. The yields and quality characteristics of recently introduced resistant cultivars now equal those of the commercial susceptible cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

The development of Raparadish (x Brassicoraphanus, 2n=38), a new crop in agriculture

Summary Raparadish, x Brassicoraphanus, the amphidiploid hybrid between Brassica rapa (syn. B.campestris) and Raphanus sativus (fodder radish) was made by Dolstra (1982). Primary hybrid plants grew vigorously, suggesting that the amphidiploid AARR might be useful as a fodder crop. Three populations of this new material were studied, with special attention to improvement of fertility and resistance to beet cyst nematode (Heterodera schachtii), whilst preserving genetic variability. For lack of progress one of the populations was abandoned after the fourth generation. The other two populations were observed through nine or ten generations. Apart from the last two generations mass selection for seed set was carried out on the basis of single plants. This led to a considerable increase in average seed production, without losing a wide variation for this trait. Thus more progress is being expected. Five cycles of mass selection for resistance to beet cyst nematodes led to a considerable increase of the level of resistance of both populations. The prospects of this new agricultural crop are discussed.     

Major genes for resistance to beet necrotic yellow vein virus (BNYVV) in Beta vulgaris

Summary Inheritance of resistance to beet necrotic yellow vein virus (BNYVV) was studied in segregating F2 and backcross families obtained from crosses between resistant plants of the sugar beet selection Holly-1-4 or the wild beet accession Beta vulgaris subsp. maritima WB42 and susceptible parents. Greenhouse tests were carried out, in which seedlings were grown in a mixture of sand and infested soil. Virus concentrations of BNYVV in the rootlets were estimated by ELISA. To discriminate resistant and susceptible plants, mixtures of normal distributions were fitted to log₁₀ virus concentrations, estimated for segregating F1, F2 and BC populations of both accessions. The hypothesis that Holly-1-4 contained one single dominant major gene was accepted. For WB42, results fitted with the hypotheses that resistance was based on either one (or more) dominant major gene(s) showing distorted segregation, or two complementary dominant genes, which are both required for resistance. Resistance from WB42 appeared to be more effective against BNYVV than resistance from Holly-1-4.This research was carried out as part of a PhD study at the Graduate School Experimental Plant Sciences (EPS), Department of Virology, Wageningen, The Netherlands     

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.     

Selection of Diploid Nematode-Resistant Sugar Beet from Monosomic Addition Lines

Monosomic sugar beet/wild beet addition lines (2n = 19) with full resistance against the beet cyst nematode have been characterized in different ways. Within the B. procumbens and B. webbiana addition lines three groups could be classified according to their isozymes pattern, growth habit, transmission rate, and resistance level. It is assumed that B. procumbens and B. webbiana each possess three different chromosomes which carry genes for nematode resistance. In the offspring of the addition lines diploid translocation types appear at very low frequencies, Isozyme pattern or growth type of the resistant plants were used for selecting diploid types in the offspring of monosomic addition lines. Effective selection could be made in progenies of susceptible sugar beets pollinated by addition lines because the pollen transmission of the alien chromosome is very low. Using these methods 7 nematode-resistant sugar beet lines could be selected. The transmission rates of the resistance gene ranged from 70.6% to 100%. Threw heterozygous progenies showed a 1:1 segregation indicating monogenic dominant inheritance of resistance. The level of resistance was as high as in the addition lines.     

Resistance to Heterodera schachtii in patellares section of the genus Beta

Summary Fifty-two accessions of the section Patellares wild beet (including 26 Beta patellaris Moq., 13 B. procumbens Chr. Sm and 13 B. webbiana Moq.) and 14 progeny families were selected and tested against sugarbeet cyst nematode, Heterodera schachtii Schm. All Patellares species tested were highly resistant, but not immune, to the development of H. schachtii, after infection. This is the first report that mature female nematodes developed in the roots of B. webbiana plants. The occasional development of nematode cysts in roots of juvenile wild beets was, however, not a heritable genetic factor.Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Assessing fitness parameters of hybrids between weed beets and transgenic sugar beets

Herbicide resistance is a desired trait for sugar beet (Beta vulgaris L.) production because it is a low‐competitor crop that requires careful weed management. However, gene flow to weed beet (also B. vulgaris) could jeopardize the weed control strategy by causing the emergence of resistant weed beets; it could also lead to further adaptation of weed beet as a troublesome weed for other crops by selecting more competitive plants. To evaluate the hazard that such a selection process represents, apart the herbicide resistance, we investigated the morphology and reproduction of progeny of weed beets having inherited a herbicide resistance gene. First‐generation resistant weed beet exhibited traits likely counter‐selected. But such crop traits rapidly disappeared with backcrossing to weedy relatives: no biologically relevant difference was noted between resistant and susceptible near‐isogenic siblings in the various experiments. In the absence of resistance selection, our data indicate low chances for weed beet to evolve more competitive forms than present weed beet populations. However, they also suggest that there is no fitness cost limiting transgenes spread.     

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.     

Origin of the ‘Weisse Schlesische Rübe’ (white Silesian beet) and resynthesis of sugar beet

Summary Sugar beet-besides fodder beet, red beet, and chard-belongs to Beta vulgaris L. After it had been confirmed that the sugar of Beta beet is chemically identical with cane sugar, ACHARD started experiments on the production of sugar from fodder beet. He noticed that conical white beets seemed to have the highest sugar content. This first sugar beet, the Weiße Schlesische Rübe, is considered the ancestor of all sugar beets of today. It has been, and continues to be supposed that it had originated from crossings between typical fodder beet and chard. Hints in the literature about possibilities to resynthesize sugar beet by crossing fodder beet with chard were confirmed in the author's own trials; the F₂ from the crossing fodder beet Rote Walze x chard Lukullus segregated forms and colour variants largely corresponding to sugar beet. Such new sugar beets are not only important from a theoretical point of view; breeders are interested in new types, too. The synthesis of sugar beet is interpreted from a genetic point of view.     

Sources of resistance to diseases of sugar beet in related <Emphasis Type="Italic">Beta</Emphasis> germplasm: I. Foliar diseases

Resistance to four foliar diseases of sugar beet (Beta vulgaris ssp. vulgaris), virus yellows caused by Beet mild yellowing virus (BMYV) and Beet yellows virus (BYV), powdery mildew (Erysiphe betae) and Cercospora leaf spot (Cercospora beticola), was assessed in up to 600 accessions of closely related wild and cultivated Beta species. Most accessions were from the Section Beta, a taxon containing types most closely related to, and sexually compatible with, sugar beet and therefore most valuable for use in crop improvement. Between 1–12% of accessions were highly resistant (resistance scores of 2 on an international standardised resistance scale of 1–9) to these diseases. These levels, however, underestimate the potential number of resistant sources available from this section as some accessions with intermediate mean resistance scores contained a significant proportion of highly resistant plants within segregating populations. Variation in resistance to all diseases except BYV was observed within the Section Beta. Much higher levels of resistance were observed, and more frequently, in more distantly related sections of the genus Beta. Accessions of the Section Corollinae were highly resistant to both viruses (>62% of accessions tested), but less so to Cercospora (15%) and they were very susceptible to powdery mildew. Section Procumbentes accessions were highly resistant to BMYV and Cercospora (100%) but less so to powdery mildew (50%) and BYV (20%). However, sexual incompatibility between these sections and sugar beet make utilisation of these sources impractical using conventional breeding methods.     

Assessment of the degree of genetic variation in beet based on RFLP analysis and the taxonomy of Beta

Summary Clones derived from Beta vulgaris and Beta maritima were assayed for their ability to detect restriction fragment length polymorphisms (RFLPs) in different beet accessions. The clones able to detect polymorphism were used as genetic markers to assess the degree of genetic variation existing between and within different species of the genus Beta. The data support the current taxonomy of the Beta vulgaris section, while the great genetic similarity found between Beta webbiana and Beta procumbens indicates that they could belong to the same species.Enough variation was found between Beta vulgaris cultivars, allowing the isolation of a sufficient number of genetic markers for the construction of detailed genetic maps.     

The production of alien monosomic additions in Beta vulgaris as a source for the introgression of resistance to beet root nematode (Heterodera schachtii) from Beta species of the section Patellares

Summary Experiments were carried out for adding the chromosome carrying resistance to beet root nematode (Heterodera schachtii) from the wild Beta species of the section Patellares (B. procumbens, B. webbiana and B. patellaris) to the genome of B. vulgaris. Preliminary experiments indicated that crosses between the wild species and B. vulgaris cultivars of the mangold type yielded on average more viable F₁ hybrids than crosses with sugar and fodderbeet. However, crossability varied strongly between individual parental combinations. It was concluded that most types of B. vulgaris can be hybridized with the wild species of the section Patellares if a sufficient number of pair-crosses is made. Crosses between diploid cultivars or species of the section Vulgares and diploid wild species of the section Patellares yielded many hybrids which, however, were highly sterile. From crosses between tetraploid B. vulgaris and the wild species a great number of viable allotriploid and allotetraploid hybrids was obtained. In the backcross progenies of allotriploid hybrids 26% alien monosomic additions occurred, of which 4.1% carried the resistance bearing chromosome of B. procumbens or B. patellaris. The programme will be continued by sereening progenies of the resistant monosomic addition plants for the occurrence of resistant disomic introgression products.     

Natural variation within the genus Beta and its possible use for breeding sugar beet: A review

Summary Sugar beet is a relatively young crop, which supposedly has a narrow genetic base. Natural variation occurring in primitive beet varieties and in wild Beta species has been used for breeding sugar beet. This paper reviews information on desirable characteristics in Beta germplasm and the attempts made for the introgression of such characters into commercial breeding material. After an introduction on the availability of germplasm and the possibilities of hybridisation, attention is focussed on the mating system (especially male sterility), on morphological and physiological characteristics, including yield and sugar content, and on resistances to diseases and pests.     

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic <Emphasis Type="Italic">cry1Ab</Emphasis> gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F₁ plants obtained through crossing of T₀ plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.     

Development of bio-assays and screening for resistance to beet armyworm (Spodoptera Exigua Hübner) in Allium cepa L. and its wild relatives

The beet armyworm (Spodoptera exigua Hübner)is the most important pest in tropical Alliumcultivations. All shallot (Allium cepa L. group Aggregatum) cultivars are susceptible to this pest. Therefore accessions from three wild Alliumspecies, namely A. galanthum Kar. et Kir., A. fistulosum L. and A. royleiStearn, next to A. cepa L. were used to screen for resistance. First of all, a reliable bio-assay had to be developed. To this end transparent plastic cages with in total 5 plants of one accession per cage were placed on per lite in a heated greenhouse. Five 3-day old larvae were inoculated on each plant. Eight days after inoculation the number of surviving larvae per cage and the mean fresh weightper larva was determined. The lowest larval survival (36%) was found on A. roylei. This was not, however, significantly different from other Allium accessions. Significant differences were found in the fresh weight per larva fed on different Allium accessions. The larvae survived on A. roylei had a very low fresh weight (10.3 mg per larva), while those on an accession of A. fistulosum had the highest fresh weight (45.1 mg per larva). The larval fresh weight on A. roylei was lower than all the other accessions except from the tropical shallot cultivar Bawang Bali. To check whether or not a toxic compound was involved in the resistance present in A. roylei, tenaccessions from four Allium species were screened. Five 3-day old larvae were inoculated on regularly replaced leaf material of each accession ofAllium species. No significant differences were found in mean fresh weight per larva and mean survival of larvae among different accessions. There were also no significant differences in pupal weight and developmental time. All larvae became pupae 10 days after inoculation. The data indicate that there is no toxic compound present in A. roylei. These results are underlined by the observation in the greenhouse bio-assay that A. roylei plants were equally damaged by the beet armyworm compared to otherAllium species. The results obtained so far therefore suggest that introduction of resistance to S. exigua via the exploitation of variation for resistance to the beet armyworm in A. roylei is unclear and that genetic engineering using Cry sequences could provide a way forward. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reduced titer of BNYVV in transgenic sugar beets expressing the BNYVV coat protein

Summary Rhizomania is a disease of sugar beet caused by the furovirus beet necrotic yellow vein virus (BNYVV). Coat protein mediated resistance has been reported for a number of viral diseases. This approach to virus resistance was therefore attempted for control of rhizomania. Two constructs of the coat protein gene of BNYVV were introduced into sugar beet by Agrobacterium-mediated transformation. The mRNA level was estimated to be 0.01% of the poly A⁺ RNA. Expression of the coat protein gene was under the detection limit of our western blotting protocol i.e. below 0.01 g/50 g (0.02% of the total soluble protein). One transformation event per construct was tested in a greenhouse assay and in rhizomania infested soil in a field trial. In the greenhouse assay, transgenic plants showed a strong reduction of virus multiplication when compared to non-transgenic plants. This result was confirmed in the field trial, where a significant difference in virus multiplication was shown between plants with and without the coat protein gene.