Transgenic Sugar Beet Plants Research Articles

Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.

Comparative proteome analyses of rhizomania resistant transgenic sugar beets based on RNA silencing mechanism

ABSTRACT Rhizomania is an economically important disease of sugar beet, which is caused by Beet necrotic yellow vein virus (BNYVV). As previously shown, RNA silencing mechanism effectively inhibit the viral propagation in transgenic sugar beet plants. To investigate possible proteomic changes induced by gene insertion and/or RNA silencing mechanism, the root protein profiles of wild type sugar beet genotype 9597, as a control, and transgenic events named 6018-T3:S6-44 (S6) and 219-T3:S3-13.2 (S3) were compared by two-dimensional gel electrophoresis. The accumulation levels of 25 and 24 proteins were differentially regulated in S3 and S6 plants, respectively. The accumulation of 15 spots were increased or decreased more than 2-fold. Additionally, 10 spots repressed or induced in both, while seven spots showed variable results in two events. All the differentially expressed spots were analyzed by MALDI-TOF-TOF mass spectrometry. The functional analysis of differentially accumulated proteins showed that most of them are related to the metabolism and defense/stress response. None of these recognized proteins were allergens or toxic proteins except for a spot identified as phenylcoumaran benzylic ether reductase, Pyrc5, which was decreased in the genetically modified S6 plant. These data are in favor of substantial equivalence of the transgenic plants in comparison to their related wild type cultivar since the proteomic profile of sugar beet root was not remarkably affected by gene transfer and activation RNA silencing mechanism.

Transgenic sugar beet tolerant to imidazolinone obtained by Agrobacterium-mediated transformation

Sugar beet is highly sensitive to imidazolinone herbicides thus rotational restrictions exist. In order to develop imidazolinone tolerant sugar beets als gene from Arabidopsis thaliana encoding acetolactate synthase with S653N mutation was used for genetic transformation. Transgenic sugar beet plants were obtained by Agrobacterium-mediated transformation of aseptic seedlings using vacuum-infiltration. The efficiency of genetic transformation was 5.8%. RT-PCR analysis of obtained plants revealed accumulation of specific als transcript. The resistance to imidazolinone was proved for developed transgenic sugar beet plants in vitro and in greenhouse conditions after spraying with imazethapyr (Pursuit, BASF).

Ambivalent effects of defective DNA in beet curly top virus-infected transgenic sugarbeet plants

Beet curly top virus (BCTV) limits sugarbeet production considerably. Previous studies have shown that infections are associated with the generation of defective DNAs (D-DNA) which may attenuate symptoms. Transgenic sugarbeet lines were established carrying a partial direct repeat construct of D-DNA in order to examine whether they are useful as a means of generating tolerance against BCTV. Thirty four independent transgenic lines were challenged. Viral full-length and D-DNAs were monitored by polymerase chain reaction (PCR) or rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP). The differential accumulation of both DNA species was compared with symptom severity during the course of infection. RCA/RFLP allowed the discrimination of two D-DNA classes which were either derived from the transgenic construct (D 0) or had been generated de novo (D n ). The statistical analysis of the results showed that the presence of D 0-DNA correlated with increased symptom severity, whereas D n -DNAs correlated with attenuated symptoms.

Transposition of the maize transposable element dSpm in transgenic sugar beets

Transgenic sugar beet plants carrying maize Spmn/dSpm transposable elements system have been constructed. Heterologous system of maize transposable elements Spm/dSpm was active in transgenic sugar beets that permits transposon-based gene tagging and obtaining of marker-free transgenic sugar beet.

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic cry1Ab 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.

Taproot promoters cause tissue specific gene expression within the storage root of sugar beet

The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.

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.

Monitoring the spread of recombinant DNA from field plots with transgenic sugar beet plants by PCR and natural transformation of Pseudomonas stutzeri.

Previous studies had shown that recombinant DNA can be detected for several months in soil after the deposition of litter from transgenic (tg) plants. Here we show by PCR monitoring of field releases of tg sugar beet plants that during the growth of the plants the soil close to the plants and also plant material contains recombinant DNA, in the form of extracellular molecules. Surprisingly, the monitoring also revealed the presence of tg DNA in many field plots (30-70%) in which tg plants were never grown. These studies and the further monitoring during other tg sugar beet release experiments by PCR and a novel bioassay (measuring the transforming potential of recombinant DNA for Pseudomonas stutzeri) indicated that recombinant DNA was only detectable in the surface soil of field plots and their vicinity where flowering of the tg beet plants was allowed. Recombinant DNA was found in soil at a distance of 50 m from pollen-producing plants surrounded by a strip with hemp plants as a containment regime. It is concluded that recombinant DNA is deposited in soil during the growth of tg sugar beets and that a major mechanism of recombinant DNA spread in the environment is the dispersal of pollen which allows recombinant DNA to persist in the field plot for at least a year.

Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugarbeet ( Beta vulgaris L.) by Agrobacterium or particle bombardment

Two different methods for sugarbeet (Beta vulgaris L.) transformation were developed, one using Agrobacterium with excised cotyledons, the other, particle bombardment of embryogenic hypocotyl callus. Transformation efficiencies averaged 0.7% for the Agrobacterium method (number of transgenic plants obtained per treated cotyledon) and about 8% for the bombardment method (number of transgenic plants obtained per plate of embryogenic callus treated). Transgenic sugarbeet plants were produced carrying genes encoding either pathogen-defense-related proteins or the reporter enzyme β-glucuronidase (GUS) under transcriptional control of stress- or wound-inducible promoters. In addition, two plants were regenerated carrying a gene associated with enhanced insect resistance, the cytokinin biosynthesis gene, fused to a patatin gene promoter from potato. Expression of the GUS gene (gusA) under the control of the tobacco osmotin promoter was wound inducible with detectable activity at 8 h and maximal activity at 72 h post-wounding.

Analysis of gene inheritance and expression in hybrids between transgenic sugar beet and wild beets

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets (Beta vulgaris ssp. maritima) has to be considered. In this study the transfer of BNYVV‐ (beet necrotic yellow vein virus) resistance and herbicide‐tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.

Activity of β-glucuronidase in Root Tips of Different Types of Transgenic Sugar Beet Plants

Expression of the β-glucuronidase (GUS) reporter gene driven by the CaMV 35S, rolC, nos and mas promoters was assessed in the tips of 12 independent clones of transgenic sugar beet (Beta vulgaris) roots. Three questions were addressed: 1) expression pattern specific for a given promoter, 2) expression pattern variability, and 3) relationship between gene expression and cell differentiation. Characteristic patterns of tissue-specific expression were distinguished for each promoter. Striking differences, however, were found between some clones, bearing the same construct. Statistical analysis of the pattern variability proved that the variability is significantly lower within the construct than between constructs. rolC-GUS clones exhibited the lowest and CaMV 35S clones the highest pattern variability. Comparisons between the four promoters showed consistent GUS activity in areas playing a key role in tissue determination (the elongation zone) where cells switch from frequent mitosis and mostly isodiametrical growth, typical for the promeristem, to rapid elongation and differentiation. All of the clones were highly GUS-positive in the elongation zone of stele. Activity was commonly localised in the stele of the maturation zone for CaMV 35S, rolC and mas-GUS clones. CaMV 35S-GUS clones were highly active in the promeristem.

The effect of exogenously-applied phytohormones on gene transfer efficiency in sugarbeet ( Beta vulgaris L.)

Cellular competence for regeneration and transformation has been monitored in a recalcitrant crop, i.e. sugarbeet ( Beta vulgaris L.). Explants for Agrobacterium inoculation were taken from the transition zone between hypocotyl and cotyledonary petioles. De novo generation of adventitious shoots was very efficient (60%–70%) from these cotyledonary node explants. Regeneration could not be further stimulated by manipulation of cytokinin type or concentration. The mode of regeneration was direct, without a callus phase. Dedifferentiation, as induced by a preculture period on 2,4-D, gave significantly higher gene transfer frequencies, but regeneration was irreversibly impaired. NAA mimicked the effect of 2,4-D on gene transfer. However, explants retained their regenerative capacity upon pretreatment with NAA. Using the procedure described here, transgenic sugarbeet plants were obtained after selecting on 250 mg/l kanamycin at a frequency of 1%.