Production of transgenetic sugarbeet (Beta vulgaris L.) plants resistant to phosphinothricin

A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) with vacuum infiltration has been developed. Aseptic 3-weeks old etiolated seedlings of two diploid O-type sugarbeet lines (KS3 and KS7) have been used for genetic transformation. Transgenic sugarbeet plants carrying the reporter beta-glucuronidase gene have been selected for their resistance to glufosinate ammonium herbicide. Integration of transgenes into sugarbeet genome was confirmed with GUS assay and PCR using primers for bar and gusA genes.     

Species of small DNA molecules found in mitochondria from sugarbeet with normal and male sterile cytoplasms

Summary Mitochondrial DNA and RNA were isolated from a range of normal and cytoplasmically male sterile sugarbeet varieties and breeding lines. When these nucleic acids were analysed by electrophoresis on agarose gels it was found that mitochondria from normal sugarbeet contain DNA species of sizes 1.3 kilobase pairs (kbp), 1.4 kbp and one of two other species of sizes 1.45 kbp and 1.5 kbp, in addition to their DNA of relatively much higher molecular weight. In contrast mitochondria from cytoplasmically male sterile sugarbeet contained only one of these DNA species, that of 1.5 kbp. Treatment with DNaseI, RNase and nuclease S1 showed that these species consisted of supercoiled circular DNA. It is not known whether the lack of the two smaller DNA species causes cytoplasmic male sterility, or whether the two traits are associated by chance.In addition it was found that mitochondria from some individual sugarbeets contained one or more types of high molecular weight RNA molecules which were probably double stranded. Also mitochondria from some sugarbeet lines and varieties contained a series of DNA molecules with molecular weights in the range 2 to 10 kbp. Neither these DNA molecules nor the RNA molecules were apparently correlated with cytoplasmic male sterility.     

Attempts to define and mimic the effects of pollen on the development of lesions caused by Phoma betae inoculated onto sugarbeet leaves

Expanding lesions resulted when conidia of Phoma betae Frank, mixed with rye pollen, were inoculated on to sugarbeet leaves by a standard technique. Conidia without pollen generally caused non-expanding necrotic spots; these could be made to spread later by covering them with pollen or orange juice, but not with water. Germ-tube growth was quicker on water agar than on sugarbeet leaves. Pollen extract stimulated germ-tube growth on leaves 10 h after inoculation and resulted in the production of knots of hyphae overlying areas where intercellular hyphae could be discerned and where expanding lesions developed. Some inorganic salts mimicked the stimulatory effect of pollen on germtube growth on agar slides, but only a mixture of hexose sugars with boric acid reproduced the effect of pollen on both numbers and size of expanding lesions caused by P. betae on sugarbeet leaves, and numbers of expanding lesions caused by Botrytis cinerea Pers. ex Fr. on bean leaves.     

Development of Heterodera schachtii on Large Rooted Crop Plants and the Significance of Root Debris as Substratum for Increasing Field Infestations

Heterodera schachtii developed to maturity and reproduced on the lateral roots of defoliated sugarbeet which were buried to a depth of 2.5 cm in sterilized soil and inoculated with cysts. Nematodes did not develop on detached lateral roots or on roots of young defoliated beets which did not have a large tap root. The storage roots of large rooted plants were sliced, placed in small jars, inoculated with cysts, covered with moist granulated agar or soil and incubated at 24°C 12-62 days. The sugarbeet nematode developed in root slices of sugarbeet, red table beet, icicle and globe radish, turnip and rutabaga. Only a few males developed on slices of potato tubers. Neither males nor females developed on root slices of carrot, salsify or parsnip. H. schachtii also developed on the cut surfaces of growing sugarbeet and radish.     

An integrated approach for the evaluation of biological control of the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus,by means of seed inoculants

Rhizomania is an extremely severe sugarbeet disease caused by the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus (BNYVV). A relatively small number of recently introduced sugarbeet cultivars characterized by a high tolerance to rhizomania are available on the market. An integrated approach was therefore developed using Pseudomonas fluorescens biological control agents (BCAs) in order to improve yield performance of cultivars characterized by a medium tolerance to the disease. A genetically modified biological control agent, Pseudomonas fluorescens F113Rif (pCUGP), was developed for enhanced production of the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) and lacking an antibiotic resistance marker gene, making the strain suitable for field release. The ability of synthetic Phl and P. fluorescens F113Rif (pCUGP) to antagonize the fungal vector, P. betae, was assessed in microcosm trials. Results encouraged the preparation of multiple field trials in a soil naturally infested with P. betae/BNYVV, to determine the biocontrol efficacy of P. fluorescens F113Rif (pCUGP) and to assess its impact on sugarbeet yield and quality and on the indigenous microbial population. While the colonization ability of P. fluorescens F113Rif (pCUGP) was satisfactory at sugarbeet emergence (2.5×10⁶ CFU g^–1 root), control of rhizomania was not achieved. Inoculation of sugarbeet with Pseudomonas fluorescens F113Rif (pCUGP) did not affect crop yield and quality nor affect the numbers of selected microbial populations.     

Parasitism of Nonhost Cultivars by Ditylenchus dipsaci

The alfalfa race of Ditylenchus dipsaci parasitized and caused characteristic symptoms on nonhost seedlings of sweet clover, onion, tomato, sugarbeet, and wheat in controlled growth-chamber studies. Although the nematode was unable to reproduce on any of the cultivars, it caused plant mortality ranging from 20% on sugarbeet and tomato to 100% on onion.     

Host Suitability of Rapeseed for Heterodera schachtii

Because rapeseed, especially canola, has the potential to be grown in rotation with sugarbeet in the north-central region of the United States, this study was initiated to assess its susceptibility to infection by Heterodera schachtii and to develop a screening method for Brassica germplasm. Existing methodology was adapted for growing Brassica juncea, B. napus, B. rapa, Brassica hybrids, and sugarbeet, Beta vulgaris, in H. schachtii-infested soil to count the females that developed on the roots. Cysts on sugarbeet contained a mean of 130 eggs compared with 240 for B. napus, lowest for the Brassica. Viability of eggs produced was assessed in soil planted with Brassica and sugarbeet and infested with with 0, 100, 1,000, 3,000, and 5,000 eggs to count resulting females and cysts. Number of females (y) was related linearly to infestation rate (x) by the regression equations y = 2.82 + 0.07(x) for the Brassica lines (R² = 0.79; P < 0.001) and y = 0.43 + 0.04(x) for sugarbeet (R² = 0.69; P < 0.007). These data indicated the potential for H. schachtii population increase if the two crops are used in rotation. All of the 111 germplasm lines tested were susceptible. The methodology developed during this research would benefit attempts to develop rapeseed cultivars resistant to H. schachtii.     

A Simulation Model of Heterodera schachtii Infecting Beta vulgaris

A simulation model of a single sugarbeet, Beta vulgaris L., plant infected by the sugarbeet cyst nematode, Heterodera schachtii Schmidt, was developed using published information. The model is an interactive computer simulation programmed in FORTRAN. Given initial population densities of the nematode at planting, the model simulates nematode population dynamics and the growth of plant tap and fibrous roots. The driving variable for nematode development and plant growth is temperature.     

Evaluation of AFLP in Beta

 AFLP markers were evaluated for their usefulness in the genetic analysis of sugarbeet and wild Beta species. Accessions of ten different sugarbeet breeding lines and five wild beets were screened using 256 primer combinations. Of the 11 309 bands investigated, 96.4% were polymorphic among the accessions. A strong positive correlation was found between the number of polymorphisms and AT content of the selective bases of the primer combinations. Random subsets of primer combinations were used to produce genetic distance trees. Permutation tests showed that, for the wild beets, 500 AFLP bands sufficed to obtain the best topology of the tree with a probability at any given node of more than 99%. Ten times as many bands were necessary to obtain support values of the same order of magnitude for the sugarbeet lines. The reproducibility of AFLP for seven primer combinations was investigated by repeated analysis of all steps from DNA isolation to data scoring. For 5088 comparisons, the overall reproducibility was 97.6%. Robustness to genotyping errors was investigated by including an artificial F₁ (1 : 1 DNA mixture) of two sugarbeet lines in the screen for polymorphisms. For the 3160 cases of polymorphism between the two lines, 0.2% genotyping errors were found. The general reliability and usefulness of AFLP markers are discussed in relation to the results obtained. Received: 18 May 1998 / Accepted: 28 October 1998     

Production of transgenic sugarbeet plants (Beta vulgaris L.) using Agrobacterium rhizogenes

Normal phenotype sugarbeet plants transformed with Agrobacterium rhizogenes were produced using direct regeneration from explants without hairy root phase. Kanamycin resistant plants and Ri-roots carrying the genes of neomycin phosphotransferase II and b-glucuronidase have been obtained. Integration of transgenes into sugarbeet genome was confirmed with GUS-assay and PCR using primers for the introduced genes.     

Native β-glucuronidase activity in sugarbeet (Beta vulgaris)

β-Glucuronidase (EC 3.2.1.31) activity, initially thought absent from plants, has been found in a number of plant families. During an analysis of Agrobacterium -mediated transformation of sugarbeet ( Beta vulgaris L.), significant glucuronidase activity was observed in control (non-transformed) tissues when the fluorogenic substrates 4-methylumbelliferyl-β- d -glucuronic acid, resorufin glucuronic acid and 3-carboxyum-belliferyl-β- d -glucuronic acid were used to quantify β-glucuronidase activity under standard protocol conditions. Similarly, the colorigenic substrate p -nitrophenyl-β- d -glucuronide was hydrolyzed by this sugarbeet-derived glucuronidase. Biochemical and immunological data are presented to indicate significant differences between sugarbeet-derived glucuronidase and that from Escherichia coli (EC 3.2.1.31) encoded by gusA . These differences provide means of distinguishing between the two activities in extracts that contain a mixture of both. Use of X-glue, the substrate utilized in histochemical localizations of glucuronidase activity, gave no reaction product (i.e., indigo precipitate) at pH 7.0. However, at pH 3.0, 4.0 and 5.0 formation of the indigo precipitate was evident within 1 h at 37°C in sugarbeet callus and by 4 h in leaves and petioles. The specific activity of sugarbeet glucuronidase was observed to be strongly pH dependent, with an optimum near pH 4.0. The use of various β-glucuronidase assay techniques as applied to transformation of sugarbeet is discussed.     

Sugarbeet culture and mormon economic development in the Intermountain West

Beta vulgaris L. played an important economic role in Mormon settlements from 1852 to 1979. The introduction of the sugarbeet from Europe supplied not only a source of sugar and income, but also a sense of dignity and independence to early Mormon pioneers seeking refuge from intense religious persecution. As the major cash crop in early Mormon settlements, cultivation of sugarbeet directly provided security for farmers, and indirectly the economic foundation for workers in many other areas. However, in the last three decades, changes in world and U.S. sugar prices have rendered sugarbeet cultivation in some areas of the Intermountain West economically untenable, and the crop has been commercially abandoned.     

Plasma membrane ATPase of sugarbeet

A membrane fraction enriched with magnesium-dependent ATPase activity was isolated from sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI and sucrose density gradient centrifugation. This activity was inhibited by vanadate, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol, but was insensitive to molybdate, azide, oligomycin, ouabain, and nitrate, suggesting enrichment in plasma membrane ATPase. The enzyme was substrate specific for ATP, had a pH optimum of 7.0, but showed little stimulation by 50 mM KCl. The sugarbeet ATPase preparation contained endogenous protein kinase activity which could be reduced by extraction of the membranes with 0.1% (w/v) sodium deoxycholate. Reduction of protein kinase activity allowed the demonstration of a rapidly turning over phosphorylated intermediate on a M_r 105000 polypeptide, most likely representing the catalytic subunit of the ATPase. Phosphorylation was magnesium dependent, sensitive to diethylstilbestrol and vanadate but insensitive to oligomycin and azide. Neither the ATPase activity nor phosphoenzyme level were affected by combinations of sodium and potassium in the assay. These results argue against the presence of a synergistically stimulated NaK-ATPase at the plasma membrane of sugarbeet.     

Impact of salinity stress on soil-borne fungi of sugarbeet

The pathogenicity of Egyptian and German isolates of soil-borne root rotting fungi to seedlings of three cultivars of sugarbeet in presence or absence of different concentrations of either NaCl or CaCl₂ were studied under greenhouse conditions. In the absence of salt treatments Egyptian isolates ofRhizoctonia solani were most virulent on all sugarbeet cultivars followed bySclerotium rolfsii andFusarium oxysporum f. sp.betae, the latter proved to be a weak pathogen. The results also revealed that the German isolates ofSclerotinia sclerotiorum were pathogenic to all sugarbeet cultivars studied, whileBotrytis cinerea was only a weak pathogen. However, the presence of salts, NaCl or CaCl₂, in different concentrations seemed to cause alterations in such pathogenicity.     

Habituation in sugarbeet callus: auxin content, auxin protectors, peroxidase pattern and inhibitors

A GC-ECD titration of IAA in normal auxin (2,4-D)-requiring and auxin-independent (habituated) sugarbeet callus revealed an equal amount in both tissues. A comparison of the content and pattern (through starch gel electrophoresis) of soluble, membrane and wall peroxidases indicated that normal tissues contained a higher level of isoperoxidases. Normal tissues also were found to contain higher levels of peroxidase inhibitors and auxin protectors. The hypothesized peroxidase-mediated higher rate of auxin destruction in normal sugarbeet callus is supposed to be counterbalanced by the 2,4-D-controlled auxin protectors.     

Cell selection and inheritance of imidazolinone resistance in sugarbeet (Beta vulgaris)

 Sugarbeets are sensitive to imidazolinone herbicide residues applied to rotational crops. Two imidazolinone-resistance (IMI-R) sugarbeet traits were developed by somatic cell selection to overcome rotation restrictions for sugarbeets where imidazolinones have been applied. Sir-13 is an IMI-R/SU-S (sulfonylurea-sensitive) variant selected from an imidazolinone-sensitive (IMI-S) sugarbeet clone, REL-1. A second variant, 93R30B, resistant to imidazolinone as well as to sulfonylurea herbicides (IMI-R/SU-R), was selected from a plant homozygous for a previously described sulfonylurea-specific resistance trait, Sur (IMI-S/SU-R). The IMI-R alleles (Sir-13 and 93R30B) were found to be corresponding allelic variants at the same ALS locus and both were tightly associated with the Sur allele. Each resistant allele is dominant to the sensitive wild-type allele; however, incomplete dominance is shown among resistance alleles. Diploid sugarbeet contains a single ALS gene copy, limiting the ability to stack these resistance traits in the same plant by traditional breeding. Received: 1 May 1997 / Accepted: 30 June 1997     

Factors Affecting the Biology and Pathogenicity of Heterodera schachtii on Sugarbeet

A direct relationship exists between soil temperature and Heterodera schachtii development. The average developmental period of two nematode populations from Lewiston, Utah, and Rupert, Idaho, from J2 to J3, J4, adult, and the next generation J2 at soil temperatures of 18-28 C were 100, 140,225, and 399 degree-days (base 8 C), respectively. There was a positive relationship (P < 0.05) between nematode Pi, nematode generations, and sugarbeet yields. The greatest sugarbeet growth inhibition (87%) occurred when sugarbeets were exposed to a Pi of 12 eggs/cm³ soil for five generations (1,995 degree-days), compared with a 47% inhibition when plants were exposed to the same Pi for two generations. There was a negative correlation (P < 0.05) between the Pi, Pf, and sugarbeet yield for each population threshold. The smaller the Pi, the greater the sugarbeet yields and the greater the Pf. Root yields were 80 and 29 t /ha and Pf were 8.4 and 3.6 eggs/cm³ soil when sugarbeet seeds were planted at Pi of 0.4 and 7.9 eggs/cm³. respectively, at a soil temperature of 8 C. The number of years rotation with a nonhost crop required to reduce the nematode population density below a damage threshold level of 2 eggs/cm³ depends on the Pi. A Pi of 33.8 eggs/cm³ soil required a 5-year crop rotation, whereas a Pi of 8.4 eggs/cm³ soil required a 2-year crop rotation.